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BpCCR1正义链及反义链对7年生盆栽白桦木质素的影响及优良株系选择

张嫚嫚, 刘宝光, 顾宸瑞, 王楚, 陈肃, 姜静, 刘桂丰

张嫚嫚, 刘宝光, 顾宸瑞, 王楚, 陈肃, 姜静, 刘桂丰. 转BpCCR1正义链及反义链对7年生盆栽白桦木质素的影响及优良株系选择[J]. 北京林业大学学报, 2019, 41(6): 86-95. DOI: 10.13332/j.1000-1522.20180412
引用本文: 张嫚嫚, 刘宝光, 顾宸瑞, 王楚, 陈肃, 姜静, 刘桂丰. 转BpCCR1正义链及反义链对7年生盆栽白桦木质素的影响及优良株系选择[J]. 北京林业大学学报, 2019, 41(6): 86-95. DOI: 10.13332/j.1000-1522.20180412
Zhang Manman, Liu Baoguang, Gu Chenrui, Wang Chu, Chen Su, Jiang Jing, Liu Guifeng. Effects of transgenic sense and antisense of BpCCR1 on 7-year-old potted birch and selection of excellent lines[J]. Journal of Beijing Forestry University, 2019, 41(6): 86-95. DOI: 10.13332/j.1000-1522.20180412
Citation: Zhang Manman, Liu Baoguang, Gu Chenrui, Wang Chu, Chen Su, Jiang Jing, Liu Guifeng. Effects of transgenic sense and antisense of BpCCR1 on 7-year-old potted birch and selection of excellent lines[J]. Journal of Beijing Forestry University, 2019, 41(6): 86-95. DOI: 10.13332/j.1000-1522.20180412

BpCCR1正义链及反义链对7年生盆栽白桦木质素的影响及优良株系选择

基金项目: 国家重点研发计划课题资助(2017YFD0600603)
详细信息
    作者简介:

    张嫚嫚。主要研究方向:林木遗传育种。Email:2450772378@qq.com 地址:150040 黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林学院

    责任作者:

    刘桂丰,教授,博士生导师。主要研究方向:林木遗传育种。Email:liuguifeng@126.com 地址:同上

  • 中图分类号: S722

Effects of transgenic sense and antisense of BpCCR1 on 7-year-old potted birch and selection of excellent lines

  • 摘要:
    目的肉桂酰辅酶还原酶(Cinnamoyl-CoA Reductase,CCR)是催化木质素合成特异途径中的第一个限速酶。通过测定转基因株系和野生型株系(WT)的木质素和单体含量,探究转BpCCR1基因正义链和反义链对白桦木质素含量的影响,进而筛选出转基因优良株系。
    方法以获得的7年生白桦转BpCCR1正、反义链株系为试验材料,采用PCR及qRT-PCR技术分别对目标基因的稳定性及表达量进行检测,采用改进的Klason法及液相色谱法分别对木质素含量及单体含量进行测定,采用硝酸-氯酸钾法和排水法分别对木纤维长和宽及基本密度进行测定,并调查树高及胸径,以此来分析转BpCCR1正、反义链对白桦上述性状的影响。
    结果PCR检测表明,5个转正义链株系及14个转反义链株系的目标基因均为阳性;qRT-PCR分析显示,BpCCR1基因不但在转正义链株系中上调表达,而且在转反义链株系中也呈上调表达。转正、反义链白桦株系木质素含量均增加,其中10个转反义链株系的Klason木质素和总木质素含量均值较野生型株系(WT)分别提高了7.46%和7.05%,木质素含量最高的FCR11株系较WT株系分别提高了12.26%和11.81%;转基因株系基本密度虽然有一定的变化,但无明显规律。转正义链株系的木纤维宽明显变小,5个株系均值较WT减少8.82%;而转反义链株系的木纤维长受到明显抑制,有11个株系与WT的差异达到了显著性水平(P < 0.05),其均值较WT减少12.12%。转基因株系与WT的材积差异也达到显著性水平,有11个转反义链株系的材积大于WT,7个株系达到显著性水平(P < 0.05),其平均材积生长量较WT提高77.1%。采用主成分分析法选择FCR2、FCR27和FCR33株系为优良株系。
    结论BpCCR1正义链及反义链均提高白桦木质素含量,综合树高、胸径等6个性状筛选出3个优良转基因株系。
    Abstract:
    ObjectiveCinnamoyl-CoA Reductase (CCR) is the first rate-limiting enzyme in the specific pathway for the synthesis of lignin and plays a crucial role in the biosynthesis of lignin. Measuring the lignin and monomer content of transgenic lines and wild lines (WT) aims to explore the effects of BpCCR1-sense and BpCCR1-antisense on the lignin of Betula platyphylla.
    Method7-year-old BpCCR1-sense and BpCCR1-antisense transgenic lines were selected as experimental materials. The expression of BpCCR1 in transgenic lines was determined using PCR and qRT-PCR, respectively. The lignin content and monomer content were determined by the modified Klason method and high performance liquid chromatography (HPLC), respectively. The length and width of the wood fiber and basic density were measured by the method of nitric acid-potassium chlorate and drainage, and the height (H) and diameter at breast height (DBH) of the trees were investigated to investigate the effects of BpCCR1 sense and antisense lines in B. platyphylla.
    ResultPCR analysis showed that the BpCCR1 was successfully integrated into the birch genome in 5 BpCCR1-sense transgenic lines and 14 BpCCR1-antisense transgenic lines. QRT-PCR analysis revealed that the expression of BpCCR1 was up-regulated in transgenic lines compared with wild type (WT). Lignin content of the transgenic lines was increased. There into, the average Klason lignin and total lignin content of 10 transgenic lines were respectively 7.46% and 7.05% higher than wild type. Compared with WT, FCR11 line had the highest content of average Klason lignin and total lignin, which was respectively increased by 12.26% and 11.81%. Although the wood basic density of transgenic lines had changed, while there was no obvious law. The wood fiber width of BpCCR1-sense transgenic lines was significantly smaller than WT, the average value of which decreased by 8.82% in five transgenic lines. Whereas, the wood fiber length of BpCCR1-antisense transgenic lines was restrained, and the difference between 11 lines and WT reached a significant level (P < 0.05), and the average value was 12.12% shorter than WT. The difference in volume between transgenic lines and WT also reached a significant level. The volume of 11 transgenic lines was larger than WT, and 7 lines reached a significant level, and the average volume growth was 77.1% higher than WT. FCR2, FCR27 and FCR32 lines were selected as excellent lines using principal component analysis.
    ConclusionBoth the sense and antisense of BpCCR1 can increase the lignin content of B. platyphylla, three excellent transgenic lines were selected by six characters including height and DBH.
  • 植物木质素主要含有对羟苯基丙烷(phydroxyphenyl propane,H)、愈创木基丙烷(guaiacyl,G)和紫丁香基丙烷(syringyl,S)3种单体[1],其主要存在于植物细胞的次生壁中,与纤维素和半纤维素共价结合,赋予细胞壁强度,为植物组织提供机械支持,使得植物向上生长,还能保护植株避免外界病原体的侵入[24]。木质素含量的增加可提高木材硬度和耐用性,进而提高木材的质量。然而,在生物能源转化及制浆造纸过程中,木质素含量高会增加能耗,并在处理过程中带来污染[5]。为此,在不影响植物组织机械强度的前提下,培育木质素含量低的纸浆材成为基因工程育种研究的热点之一,人们通常通过反义RNA技术抑制木质素单体合成代谢途径的部分关键酶,从而达到降低植物中木质素含量的目的。肉桂酰辅酶还原酶(Cinnamyl-CoA Reductase,CCR)一直被认为是催化3种木质素单体生物合成途径的第一个限速酶[6],多年来国内外针对不同植物的CCR基因开展了研究[7]。例如,将桉树(EucalyptusCCR基因的cDNA序列的反义链转入烟草(Nicotiana tabacum)中,转基因烟草木质素含量降低[8];在挪威云杉(Picea abies)中转入反义CCR基因,转基因株系木质素含量降低约8%,其中H型木质素单体含量显著降低,虽然转基因株系表型正常,但地径变小[9]。在干扰CCR基因的转基因番茄(Solanum lycopersicum)中,茎和叶中的木质素含量降低,果实中酚类物质总量没有增加,但是组分发生改变,相应地提高了番茄的抗氧化能力[10]。上述大多是通过抑制或干扰CCR基因的表达来降低木质素含量而开展的研究。但是,木质素除了增强树木机械强度和硬度等特性外尚有其他应用价值,例如,可用于制备生物燃料和高附加值化学品,并且是可再生资源[1]。从制浆废水中提取的木质素及其衍生物在农业、石油化工、水泥及混凝土工业、塑料和高分子材料等方面有着广泛的应用[11]。因此,树木中过表达CCR基因对木质素含量的提高具有重要的生产实践意义。

    白桦(Betula platyphylla)是珍贵用材及纸浆材树种,其生长快、结实量多、萌生能力强,广泛分布于我国东北、华北、西北及西南高山林区的14个省区,面积约为489.97万 hm2[12],但是白桦的木材硬度较水曲柳(Fraxinus mandshurica)、胡桃楸(Juglans mandshurica)、蒙古栎(Quercus mongolica)等树种略低,因此提高其木质素含量进而提高木材硬度是育种目标之一。反之,降低木质素含量可减少化学制浆造纸时药品对环境的污染及成本。为此,本团队前期克隆了白桦BpCCR1基因,构建了正义链和反义链表达载体,并将其转入白桦基因组中[13],获得的转基因株系已定植多年。本研究以获得的转基因株系和野生型株系(WT)为材料,探寻BpCCR1基因正义链和反义链对白桦木质素及木质素单体、木材密度及生长量等性状的影响,同时进行优良株系的筛选。

    2009年,本课题组以野生型白桦(WT)为受体,采用叶盘法进行BpCCR1正义链及反义链的遗传转化试验,获得了转正义链及反义链株系共58个,分别用CR和FCR表示。将上述转基因株系及WT定植于东北林业大学林木遗传育种试验基地中,随后在每个株系中选择生长量基本一致的3个单株移栽到花盆(45 cm × 45 cm × 30 cm)中(底部垫托盘),进行常规管理。7年生时,随机选择19个转基因株系(5个正义链、14个反义链)及对照(WT)为试验材料开展研究。

    前期获得转基因株系后分别用载体引物和基因引物以及抗性基因引物进行了检测[14],因此,本次仅对转基因株系中目标基因进行了PCR检测。取材及检测方法如下:

    (1)取材。在转基因株系及WT树冠上部的枝条上摘取从顶芽数起的第2个叶片混样进行目标基因的PCR检测,设置2次生物学重复。

    (2)检测方法。PCR检测:以含BpCCR1基因的中间表达载体的pGWB2质粒为阳性对照,以水为阴性对照对WT和19个转基因株系的基因组DNA进行PCR扩增。根据目的基因序列上游及下游序列设计引物,其中,转正义链株系的上、下游引物分别为:5′ATGGGGATTCTGGGGGCCGAAG3′、5′CTAACATTCATTGTAACAATATCGGAGTGTTC3′;转反义链株系的上、下游引物分别为:5′CTAACATTCATTGTAACAATATCGGAGTGTTC3′、5′ATGGGGATTCTGGGGGCCGAAG3′。反应体系及扩增条件参考王朔等[14]的研究。

    qRT-PCR检测:根据BpCCR1基因的蛋白编码区(CDS)序列设计qRT-PCR引物(表1),检测转基因株系中BpCCR1基因(包括转入的BpCCR1基因及内源BpCCR1基因)表达特性;根据BpCCR1基因的3′ UTR序列设计引物,检测转基因株系中内源BpCCR1基因的表达特性。qRT-PCR反应体系及扩增条件参考黄海娇等[15]的研究。BpCCR1基因相对表达量为− ΔΔCt,内源BpCCR1基因相对表达量为2− ΔΔCt

    表  1  BpCCR1基因及内源BpCCR1基因qRT-PCR引物序列
    Table  1.  qRT-PCR primer sequence for BpCCR1 gene and endogenous BpCCR1 gene
    基因名称 Gene name   正向引物(5′→3′) Forward primer (5′→3′)反向引物(5′→3′) Reverse primer (5′→3′)
    18S rRNAGAGGTAGCTTCGGGCGCAACTGCAGGTTAGCGAAATGCGATAC
    BpCCR1 AGCATGTGCGAGAACACCATCACTCATCACTCCAGCAGCCA
    内源BpCCR1 Endogenous BpCCR1CAAGAATGCAGCAGGCAGATACGAGAGAGGTGACATAAACGGCC
    下载: 导出CSV 
    | 显示表格

    (1)取材:分别选择19个转基因株系及WT株系各1株,截取地上部位15 cm ~ 35 cm处的树干,将其分上、中、下3部分,下部的6 cm木段平均分成3段用于木材密度测定,中间的10 cm木段用于木质素及木质素单体的测定,上部的4 cm木段用于纤维长宽的测定。

    (2)木质素及木质素单体测定。采用改进的Klason方法测定木质素含量[1617]。木质素单体的测定需将干燥的木粉在氢氧化钠和硝基苯混合溶液中高温高压反应,然后用HCl调整溶液pH值。将经过分解的木材样品溶液在液相色谱仪中进行测定,计算6种组分的质量,再将组成每种木质素单体的2种组分含量相加,得出样品中3种单体的含量。

    (3)木材基本密度测定。采用排水法进行测定[18]

    (4)纤维长宽测定。采用硝酸−氯酸钾法测定[1920]

    采用5 m塔尺测定树高,用游标卡尺测定地径及胸径。参考白桦的二元材积表[2122],利用测定的树高(H)和胸径(D)计算单株材积(V),V = 0.000 051 935 163D1.858 688 4 H1.003 894 1

    试验数据采用Excel 2010和SPSS 19.0软件进行分析。数据处理采用单因素方差分析法(One-way ANOVA)和Duncan’s test多重比较法检验样品间的差异显著性。

    对7年生转基因白桦株系采用PCR扩增技术检测导入BpCCR1基因的稳定性(图1),结果表明,目标基因在5个转正义链株系及14个转反义链株系均有1 089 bp的扩增谱带,说明导入的正义链、反义链仍整合在转基因株系基因组中。进一步利用qRT-PCR分析BpCCR1基因的表达特性,结果表明,BpCCR1基因的相对表达量不仅在5个转正义链株系中较WT明显提高,而且在14个转反义链株系中也有明显提高,且多数株系显著高于转正义链株系(P < 0.05)(图2)。对内源BpCCR1基因进行qRT-PCR检测,结果表明,转正义链株系中80.00%的株系、转反义链株系中85.71%的株系的内源BpCCR1基因的相对表达量显著高于WT(图3)。

    图  1  BpCCR1基因株系PCR检测
    M. DL2000;1. 阳性质粒;2. 水对照;3. 野生型(WT);4 ~ 8. 转正义链株系;9 ~ 22. 转反义链株系。M, DL2000; 1, plasmid control; 2, water control; 3, wild type (WT); 4−8, sense BpCCR1 transgenic line; 9−22, antisense BpCCR1 transgenic line.
    Figure  1.  PCR detection of BpCCR1 transgenic lines
    图  2  BpCCR1基因株系qRT-PCR检测
    WT. 野生型;CR. 转BpCCR1正义链株系;FCR. 转BpCCR1反义链株系;不同小写字母代表差异显著,P < 0.05。下同。WT, wild type; CR, sense BpCCR1 transgenic lines; FCR, antisense BpCCR1 transgenic lines; different lowercase letters represent significant differences at P < 0.05 level. The same below.
    Figure  2.  Detection of BpCCR1 transgenic lines by qRT-PCR
    图  3  转基因株系内源BpCCR1基因qRT-PCR分析
    Figure  3.  Detection of endogenous BpCCR1 transgenic lines by qRT-PCR

    测定了白桦转正义链、反义链株系与WT的Klason木质素及可溶性木质素含量,并计算出总木质素含量,方差分析表明(表2),3种木质素含量分别在转正义链、反义链株系与WT间存在显著性差异(P < 0.05)。Klason木质素及总木质素含量的多重比较结果表明,5个转正义链株系中CR15株系的Klason木质素及总木质素含量显著高于WT(P < 0.05);在14个转反义链株系中,10个株系的Klason木质素及总木质素含量显著高于WT(P < 0.05)。这10个株系的2种木质素含量均值较WT分别提高了7.46%、7.05%,19个转基因株系中FCR11株系的Klason木质素含量及总木质素含量最高,较WT分别提高了12.26%和11.81%。

    表  2  BpCCR1株系木质素含量多重比较
    Table  2.  Multiple comparisons of lignin content of BpCCR1 transgenic lines
    基因
    Gene
    株系
    Line
    木质素含量 Lignin content/%木质素单体类型 Type of lignin monomer
    Klason木质素
    Klason lignin
    酸溶性木质素
    Acid-soluble lignin
    总木质素
    Total lignin
    GSS/GH
    正义链
    Sense
    CR1523.098 ± 0.206a 0.744 ± 0.015a23.841 ± 0.192a 0.261 ± 0.002cd0.726 ± 0.002c2.779 ± 0.035cd0.012 8
    CR1322.827 ± 0.287ab0.867 ± 0.158a23.694 ± 0.160ab0.280 ± 0.006a 0.715 ± 0.006d2.552 ± 0.070e 0.004 6
    CR422.683 ± 0.392ab0.776 ± 0.012a 23.459 ± 0.399abc
    CR1122.472 ± 0.497ab0.566 ± 0.035b23.038 ± 0.506bc
    WT22.042 ± 0.337b 0.845 ± 0.046a22.887 ± 0.331c 0.259 ± 0.002cd0.736 ± 0.002b2.841 ± 0.024bc0.004 3
    CR821.284 ± 0.648c 0.879 ± 0.025a22.163 ± 0.626d 0.243 ± 0.002e 0.752 ± 0.002a3.101 ± 0.026a 0.005 0
    反义链
    Antisense
    FCR1124.745 ± 0.065a 0.844 ± 0.061abcd25.589 ± 0.091a 0.257 ± 0.001d 0.738 ± 0.001b2.873 ± 0.011b 0.005 0
    FCR824.338 ± 0.109ab 0.816 ± 0.111bcd25.154 ± 0.190ab
    FCR3624.323 ± 0.116ab 0.799 ± 0.061cde25.122 ± 0.128ab
    FCR2523.896 ± 0.500bc 0.933 ± 0.026ab24.829 ± 0.526b
    FCR523.617 ± 0.164cd0.960 ± 0.041a24.578 ± 0.204bc
    FCR1323.411 ± 0.145cd 0.763 ± 0.043de24.174 ± 0.139cd0.267 ± 0.003b 0.729 ± 0.003c2.725 ± 0.048d 0.003 7
    FCR1523.370 ± 0.049cd 0.738 ± 0.096def24.108 ± 0.145cd
    FCR2423.248 ± 0.022de 0.666 ± 0.143ef23.914 ± 0.124d
    FCR123.206 ± 0.432de 0.733 ± 0.089def23.938 ± 0.371cd
    FCR322.708 ± 0.566ef 0.898 ± 0.029abc23.606 ± 0.541de
    FCR3222.385 ± 0.426fg 0.752 ± 0.048de23.137 ± 0.395ef
    WT22.042 ± 0.337g 0.845 ± 0.046abcd22.887 ± 0.331fg0.259 ± 0.002cd0.736 ± 0.002b2.841 ± 0.024bc0.004 3
    FCR222.016 ± 0.356g 0.669 ± 0.058ef22.685 ± 0.380fg0.262 ± 0.001c 0.734 ± 0.001b2.796 ± 0.017c 0.004 4
    FCR3322.010 ± 0.497g 0.674 ± 0.037ef22.683 ± 0.534fg
    FCR2721.772 ± 0.579g 0.611 ± 0.055f 22.383 ± 0.571g
    注:“—” 代表未测定的数据;表中不同字母表示在0.05水平上差异显著;表中数据表示形式为均值 ± 标准差。下同。Notes: “—” stands for unmeasured values. Different letters mean significant difference at P < 0.05 level; data in the table are mean ± standard deviation. The same below.
    下载: 导出CSV 
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    选取木质素含量高、中、低的株系及WT,采用液相色谱法测定G型、S型和H型木质素单体含量(表2)。结果表明,S型单体含量最高、G型次之,H型极低,S/G在2.552 ~ 3.101之间。方差分析表明,各株系间的G型、S型单体含量及S/G差异达到显著水平(P < 0.05),进一步比较各株系中G型、S型单体含量及S/G,发现在转正义链株系中既有显著高于WT又有显著低于WT的株系;在转反义链的3个株系中,仅FCR13株系的G型单体含量显著高于WT,S型单体含量显著低于WT,S/G显著低于WT。

    BpCCR1基因正、反义链对白桦木纤维长、宽及长/宽等性状产生明显不同的影响(表3)。在5个转正义链株系中,木纤维长度变化不大,但宽度明显变小,宽度均值低于WT株系8.82%,故长/宽明显变大,5个株系长/宽的均值为47.24,较WT株系(40.8)提高了15.78%。在转反义链株系中,木纤维长度受到抑制,而宽度基本不变,故长/宽明显变小。其中11个株系的木纤维长度明显变短,均值较WT株系缩短12.12%;9个株系的长/宽明显变小,其均值较WT减少13.51%。

    表  3  BpCCR1株系纤维长、宽及基本密度多重比较
    Table  3.  Multiple comparisons of fiber length, width and basic density of BpCCR1 transgenic lines
    基因
    Gene
    株系
    Line
    木纤维 Wood fiber基本密度
    Basic density/(g·cm− 3)
    长 Length/μm宽 Width/μm长/宽 Length/width
    正义链 SenseCR15661.2 ± 55.5b14.4 ± 2.2b46.7 ± 3.8b0.365 5 ± 0.001 1d
    CR13657.5 ± 42.4bc14.9 ± 2.3b45.0 ± 3.8b0.392 2 ± 0.001 5b
    CR4637.0 ± 52.4c14.2 ± 2.1b45.7 ± 3.9b0.382 0 ± 0.003 9c
    CR11652.4 ± 37.9bc14.7 ± 2.5b45.4 ± 4.7b0.382 9 ± 0.008 9c
    WT650.4 ± 38.8bc16.1 ± 1.8a40.8 ± 2.3c0.381 0 ± 0.001 3c
    CR8798.3 ± 48.2a15.2 ± 1.9b53.4 ± 3.9a0.423 5 ± 0.006 7a
    反义链 AntisenseFCR11619.6 ± 38.8cd15.3 ± 1.9def41.2 ± 5.4a0.387 7 ± 0.004 5bcd
    FCR8622.2 ± 37.1cd15.4 ± 1.9def41.0 ± 5.3a0.371 9 ± 0.001 3g
    FCR36605.9 ± 43.9de16.7 ± 2.1bc36.5 ± 3.6b0.382 1 ± 0.007 9def
    FCR25562.3 ± 29.9f15.1 ± 1.5ef37.5 ± 3.9b0.376 4 ± 0.005 2fg
    FCR5587.1 ± 37.0e16.3 ± 2.8bcde37.0 ± 5.9b0.387 4 ± 0.002 1bcd
    FCR13469.6 ± 30.9h13.5 ± 1.9g35.3 ± 4.8bc0.411 0 ± 0.002 6a
    FCR15638.1 ± 31.9bc17.4 ± 2.7b37.3 ± 4.9b0.381 6 ± 0.005 3def
    FCR24610.4 ± 58.4d15.1 ± 2.2f41.1 ± 5.3a0.378 0 ± 0.000 4efg
    FCR1708.6 ± 55.4a17.3 ± 2.6b42.3 ± 4.8a0.373 3 ± 0.003 0fg
    FCR3559.2 ± 51.5f16.0 ± 2.9cdef35.9 ± 6.0bc0.379 1 ± 0.001 9defg
    FCR32544.7 ± 37.6f16.3 ± 2.1bcd33.7 ± 4.4c0.386 0 ± 0.007 9cde
    WT650.4 ± 38.8b16.1 ± 1.9cdef40.8 ± 4.3a0.381 0 ± 0.001 3def
    FCR2640.1 ± 43.9bc15.6 ± 2.9cdef42.6 ± 4.8a0.395 6 ± 0.008 0b
    FCR33609.3 ± 40.5d16.6 ± 2.0bc37.2 ± 4.5b0.385 7 ± 0.000 3cde
    FCR27496.8 ± 37.9g18.5 ± 2.2a27.2 ± 3.6d0.394 0 ± 0.005 7bc
    下载: 导出CSV 
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    木材基本密度测定结果表明,转正义链和反义链株系与WT有显著性差异(P < 0.05)(表3)。在转正义链株系中,CR8株系的密度最大(0.424 g/cm3),且显著高于WT(0.381 g/cm3),CR15株系的密度(0.366 g/cm3)显著低于WT;在转反义链株系中,FCR13、FCR2和FCR27株系的密度显著高于WT,FCR8株系显著低于WT,其他株系与WT无显著差异。木材密度的变化在转正义链和反义链株系中没有明显规律可循。

    对白桦转基因株系及WT的树高、地径、胸径和材积进行方差分析,结果表明,上述性状在转正义链和反义链株系与WT间达到差异显著性水平(P < 0.05)。多重比较发现,FCR2、FCR11和FCR8等11个株系的材积大于WT,其中7个株系达到显著性水平(P < 0.05),其平均材积生长量较WT提高77.1%(表4)。

    表  4  BpCCR1基因株系树高、地径、胸径及材积多重比较
    Table  4.  Multiple comparisons of tree height, ground diameter, DBH and volume of BpCCR1 transgenic lines
    基因 Gene   株系 Line树高 Tree height/m地径 Ground diameter/mm胸径 DBH/mm材积 Volume/cm3
    正义链 SenseCR13421.5 ± 21.5a32.4 ± 1.2ab16.3 ± 1.2b400.0 ± 32.2a
    CR4402.0 ± 11.5ab33.2 ± 1.3a17.7 ± 0.8ab447.3 ± 30.5a
    CR15388.3 ± 26.4abc30.3 ± 0.9ab18.0 ± 0.9ab446.0 ± 39.5a
    WT368.8 ± 24.8bc28.1 ± 1.6b16.7 ± 0.8b374.0 ± 20.8a
    CR11352.0 ± 10.2cd30.1 ± 0.2ab19.6 ± 1.0a473.3 ± 26.8a
    CR8310.5 ± 13.5d28.3 ± 0.9b13.8 ± 0.8c217.6 ± 31.8b
    反义链 AntisenseFCR2467.5 ± 14.5a39.1 ± 1.7a25.1 ± 1.4a993.4 ± 41.4a
    FCR11460.7 ± 16.2ab36.1 ± 2.9abc23.8 ± 0.6a889.3 ± 33.8b
    FCR8440.5 ± 12.5abc38.4 ± 2.5a20.0 ± 1.2b612.6 ± 28.9c
    FCR36432.5 ± 16.5bc31.7 ± 1.7cde17.7 ± 1.0cd483.5 ± 40.8de
    FCR33420.3 ± 6.7cd33.4 ± 0.5bcd19.7 ± 0.5b571.3 ± 20.4cd
    FCR27410.0 ± 10.0cd37.5 ± 2.2ab20.5 ± 1.3b597.4 ± 47.8c
    FCR32396.0 ± 6.0de37.1 ± 0.6ab18.8 ± 0.0bc489.6 ± 4.9de
    FCR5390.7 ± 17.8def29.6 ± 2.5de16.9 ± 0.7cde398.3 ± 27.4ef
    FCR1390.5 ± 12.5def30.2 ± 0.8de16.7 ± 0.6de391.6 ± 21.4ef
    FCR15389.0 ± 0.0def30.5 ± 0.0de17.0 ± 0.0cde400.9 ± 0.0ef
    FCR24373.0 ± 7.0ef34.3 ± 1.0abcd17.8 ± 1.3cd421.6 ± 38.1ef
    WT368.8 ± 24.8ef28.1 ± 1.6ef16.7 ± 0.8de374.0 ± 40.8f
    FCR3356.7 ± 10.8f23.9 ± 1.7f11.9 ± 0.6g191.1 ± 8.6g
    FCR25310.0 ± 5.0g30.4 ± 2.1de15.5 ± 0.5ef268.5 ± 11.8g
    FCR13306.0 ± 36.1g31.5 ± 1.3cde14.3 ± 1.6f233.9 ± 43.2g
    下载: 导出CSV 
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    对转基因株系及WT的树高、胸径及材积等6个性状分别进行了方差分析和多重比较,而优良家系的选择需要综合考虑多个性状,因此采用主成分分析法求出特征根及各性状的特征向量(表5)。因为只有2个主成分的特征根大于1,所以只选用2个主成分即可进行6个性状的综合评价(表6)。主成分的表达式为:

    表  5  特征根及标准化特征向量
    Table  5.  Characteristic roots and standardized eigenvectors
    主成分
    Main component
    特征根
    Characteristic root
    方差贡献率
    Variance contribution rate/%
    累积贡献率
    Cumulative contribution rate/%
    性状
    Trait
    因子载荷1
    Factor loading 1
    特征向量1
    Standardized eigenvector 1
    因子载荷2
    Factor loading 2
    特征向量2
    Standardized eigenvector 2
    Y12.85247.52647.526树高
    Tree height (X1)
    0.9280.549 5− 0.003− 0.002 5
    Y21.40223.37370.900胸径
    DBH (X2)
    0.9270.548 9 0.278 0.234 8
    Y30.92915.48286.382材积
    Volume (X3)
    0.9490.561 9 0.275 0.232 3
    Y40.60210.03896.420总木质素含量
    Total lignin content (X4)
    0.2370.140 3− 0.766− 0.646 9
    Y50.203 3.39199.811纤维长/宽
    Fiber length/
    width (X5)
    − 0.155 − 0.091 8 0.404 0.341 2
    Y60.011 0.189100.000 密度
    Density (X6)
    − 0.386 − 0.228 6 0.706 0.596 3
    下载: 导出CSV 
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    表  6  参试株系综合评价
    Table  6.  Comprehensive evaluation of each tested lines
    株系 
    Line 
    标准分 Standardized valueY1排名
    Rank
    Y2排名
    Rank
    X1X2X3X4X5X6
    WT− 0.442 3− 0.375 1− 0.453 0− 0.889 70.123 7− 0.369 8− 0.755 1160.205 08
    CR40.273 2− 0.064 3− 0.088 2− 0.298 70.982 9− 0.288 1− 0.001 090.320 57
    CR8− 1.698 6− 1.323 3− 1.230 8− 1.636 22.334 02.859 3− 3.448 8202.967 41
    CR11− 0.804 30.553 20.040 8− 0.733 70.933 9− 0.223 6− 0.253 0140.801 34
    CR130.693 4− 0.520 5− 0.323 7− 0.056 60.854 60.482 0− 0.283 1150.416 56
    CR15− 0.021 30.037 8− 0.094 80.095 11.150 5− 1.545 10.216 78− 0.603 413
    FCR10.025 4− 0.372 2− 0.365 30.195 10.381 9− 0.951 9− 0.185 713− 0.735 914
    FCR21.684 72.323 22.627 7− 1.097 70.432 90.735 13.315 712.447 92
    FCR3− 0.703 7− 1.912 8− 1.362 6− 0.147 3− 0.748 8− 0.510 7− 2.037 518− 1.228 618
    FCR50.029 0− 0.326 5− 0.332 00.854 3− 0.559 7− 0.719 1− 0.014 310− 1.326 220
    FCR81.102 80.672 50.734 01.449 50.154 5− 1.054 01.817 73− 1.187 816
    FCR111.537 41.911 12.109 81.897 70.189 50.142 93.295 52− 0.142 811
    FCR13− 1.795 6− 1.153 0− 1.149 70.438 4− 0.861 91.909 3− 2.561 4190.027 610
    FCR15− 0.007 0− 0.286 7− 0.319 30.370 0− 0.497 0− 0.321 9− 0.169 511− 0.742 415
    FCR24− 0.351 8− 0.020 7− 0.216 00.170 40.163 6− 0.596 3− 0.180 812− 0.464 212
    FCR25− 1.709 4− 0.776 8− 0.977 41.113 5− 0.469 80.119 4− 1.742 817− 1.214 617
    FCR270.445 60.822 40.658 3− 1.409 0− 2.277 50.615 70.936 840.846 53
    FCR320.143 90.277 50.122 0− 0.631 6− 1.131 00.010 70.312 770.122 29
    FCR330.668 30.590 30.528 3− 1.099 3− 0.523 0− 0.009 80.884 150.786 55
    FCR360.930 4− 0.056 20.091 91.415 8− 0.633 3− 0.284 00.853 86− 1.295 519
    下载: 导出CSV 
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    Y1=0.5495X1+0.5489X2+0.5619X3+0.1403X40.0918X50.2286X6 (1)
    Y2=0.0025X1+0.2348X2+0.2323X30.6469X4+0.3412X5+0.5963X6 (2)

    式中:X1X2X3X4X5、X6分别表示树高、胸径、材积、总木质素含量、纤维长/宽、密度;Y1Y2分别表示主成分1、主成分2。由公式可知Y1主要依赖于树高、胸径和材积,Y2主要依赖于总木质素含量、纤维长/宽和密度。

    根据Y1值对各家系进行综合评价,发现FCR2、FCR11、FCR8、FCR27和FCR33株系的Y1值较大,说明其树高、胸径和材积较大,即它们的生长性状较优良。根据Y2值对各家系进行综合评价,发现CR8、FCR2、FCR27、CR11和FCR33株系的Y2值最大,说明其纤维长/宽和密度较大且木质素含量较低,即材质性状优良。综合Y1Y2值进行选择,FCR2、FCR27和FCR33株系为优良株系。

    由于肉桂酰辅酶A还原酶(CCR)是木质素生物合成途径的第一个关键酶,因此,通过该基因遗传转化调控木质素含量已成为研究热点之一,并在许多转CCR基因正义链和反义链植物中获得了木质素含量增加和降低的株系,但是,在以往的研究中也出现了反常的个例。例如,有人将35S启动子驱动小麦(Triticum aestivumTaCCR1基因的正义链及反义链转入烟草中,结果发现转正义链及反义链株系中同时出现了植株矮化、木质素含量下降、木质部导管细胞壁受到破坏等现象[23]。本试验将BpCCR1基因的正、反义链转入白桦,在14个转反义链株系中有10个株系的木质素含量较WT明显增加。为此,设计特异引物分析了内源BpCCR1的表达特性,结果表明,转反义链的14个株系中有12个株系内源BpCCR1的相对表达量显著上调。有人研究发现,拟南芥(Arabidopsis thaliana)中存在正义R-loop(Sense R-loops,由基因自身转录本与其模板链形成的R-loop),也存在反义R-loop(Antisense R-loops,大多是由基因的反义转录本在正义链转录起始位点形成R-loop),R-loop是一种特殊的染色体结构,由一条RNA:DNA杂合链和一条单链DNA组成[24]。拟南芥中广泛存在一类反义长非编码RNA在正义链转录起始位点形成的R-loop,推测这类特别的R-loop在调节转录起始或控制基因转录过程中起到重要作用[24]。为此,我们推测本研究转入的反义链转录出的反义长非编码RNA在内源正义链转录起始位点形成了R-loop,进而使反义链转基因株系的内源基因上调表达,木质素含量增加。此外,生物中普遍存在与有义转录物具有互补序列的天然反义RNA,可以调节有义链基因的表达[25]。天然反义RNA调控有义链基因的作用机制呈现多样性。其中,在小鼠的转录组中发现,大部分天然反义RNA与正义基因的表达呈正相关,体现出协同表达的特征[2627]

    虽然CCR基因主要在木质素合成代谢途径中起重要作用,但研究发现转入CCR基因之后植株的其他性状也会发生一些变化。例如,在杨树(Populus tremula × P. alba)中转入CCR基因反义链后,木质素、半纤维素含量降低,纤维素含量却升高;对5年生转基因株系的木材进行化学制浆,发现制浆特性有所改善[28]。银合欢(Leucaena leucocephala)的LlCCR基因转入烟草,木质素含量减少的转反义链株系表现出全纤维素含量增加5% ~ 15%,其中AS17株系全纤维素含量增加15.53%,且生长发育迟缓,而CCR基因上调表达株系木质素含量增加,生长发育旺盛[29]。本试验中大部分BpCCR1基因上调表达的转基因株系木质素含量、树高和材积均有增加。对纤维长、宽的测定发现转入BpCCR1基因对白桦的纤维长、宽有影响,其中转入正义链使纤维宽度变小,长/宽增加;转入反义链使纤维长度变短,长/宽减少。实践证明,纤维长/宽处于35 ~ 45时适用于制浆造纸,能保证纤维良好地交织在一起[30]。在本研究中,14个转反义链株系中仅有2个株系的纤维长/宽小于35,大多数株系的长/宽处于35 ~ 45之间。

    优良家系的选择一般需要综合考虑多个性状因素,若仅靠单一性状来评价家系的优劣,会产生片面性。常用的多性状选择方法中,主成分分析法不需要主观确定权重,是根据主成分值评价各株系综合性状的优劣,能够较全面、客观地反映出各项指标的综合表现情况[3132]。本研究通过主成分分析,将参试株系的6个指标转化为2个主成分,其中主成分1主要依赖于树高、胸径和材积变化,主成分2主要依赖于总木质素含量、纤维长/宽和密度变化。根据这2个主成分,选出3个优良株系。这3个株系的综合表现明显高于其他株系,其生长性状优良,且纤维长/宽和密度较大且木质素含量较低,未来可以用于造纸和制造板材。

    BpCCR1正义链及反义链均提高白桦木质素含量。转正义链株系的木纤维宽度变小,其长/宽增加;转反义链株系材积生长量提高,木纤维长度变小,长/宽减少。利用主成分分析法对转基因株系进行综合评价,筛选出FCR2、FCR27和FCR33株系为优良株系。

  • 图  1   BpCCR1基因株系PCR检测

    M. DL2000;1. 阳性质粒;2. 水对照;3. 野生型(WT);4 ~ 8. 转正义链株系;9 ~ 22. 转反义链株系。M, DL2000; 1, plasmid control; 2, water control; 3, wild type (WT); 4−8, sense BpCCR1 transgenic line; 9−22, antisense BpCCR1 transgenic line.

    Figure  1.   PCR detection of BpCCR1 transgenic lines

    图  2   BpCCR1基因株系qRT-PCR检测

    WT. 野生型;CR. 转BpCCR1正义链株系;FCR. 转BpCCR1反义链株系;不同小写字母代表差异显著,P < 0.05。下同。WT, wild type; CR, sense BpCCR1 transgenic lines; FCR, antisense BpCCR1 transgenic lines; different lowercase letters represent significant differences at P < 0.05 level. The same below.

    Figure  2.   Detection of BpCCR1 transgenic lines by qRT-PCR

    图  3   转基因株系内源BpCCR1基因qRT-PCR分析

    Figure  3.   Detection of endogenous BpCCR1 transgenic lines by qRT-PCR

    表  1   BpCCR1基因及内源BpCCR1基因qRT-PCR引物序列

    Table  1   qRT-PCR primer sequence for BpCCR1 gene and endogenous BpCCR1 gene

    基因名称 Gene name   正向引物(5′→3′) Forward primer (5′→3′)反向引物(5′→3′) Reverse primer (5′→3′)
    18S rRNAGAGGTAGCTTCGGGCGCAACTGCAGGTTAGCGAAATGCGATAC
    BpCCR1 AGCATGTGCGAGAACACCATCACTCATCACTCCAGCAGCCA
    内源BpCCR1 Endogenous BpCCR1CAAGAATGCAGCAGGCAGATACGAGAGAGGTGACATAAACGGCC
    下载: 导出CSV

    表  2   BpCCR1株系木质素含量多重比较

    Table  2   Multiple comparisons of lignin content of BpCCR1 transgenic lines

    基因
    Gene
    株系
    Line
    木质素含量 Lignin content/%木质素单体类型 Type of lignin monomer
    Klason木质素
    Klason lignin
    酸溶性木质素
    Acid-soluble lignin
    总木质素
    Total lignin
    GSS/GH
    正义链
    Sense
    CR1523.098 ± 0.206a 0.744 ± 0.015a23.841 ± 0.192a 0.261 ± 0.002cd0.726 ± 0.002c2.779 ± 0.035cd0.012 8
    CR1322.827 ± 0.287ab0.867 ± 0.158a23.694 ± 0.160ab0.280 ± 0.006a 0.715 ± 0.006d2.552 ± 0.070e 0.004 6
    CR422.683 ± 0.392ab0.776 ± 0.012a 23.459 ± 0.399abc
    CR1122.472 ± 0.497ab0.566 ± 0.035b23.038 ± 0.506bc
    WT22.042 ± 0.337b 0.845 ± 0.046a22.887 ± 0.331c 0.259 ± 0.002cd0.736 ± 0.002b2.841 ± 0.024bc0.004 3
    CR821.284 ± 0.648c 0.879 ± 0.025a22.163 ± 0.626d 0.243 ± 0.002e 0.752 ± 0.002a3.101 ± 0.026a 0.005 0
    反义链
    Antisense
    FCR1124.745 ± 0.065a 0.844 ± 0.061abcd25.589 ± 0.091a 0.257 ± 0.001d 0.738 ± 0.001b2.873 ± 0.011b 0.005 0
    FCR824.338 ± 0.109ab 0.816 ± 0.111bcd25.154 ± 0.190ab
    FCR3624.323 ± 0.116ab 0.799 ± 0.061cde25.122 ± 0.128ab
    FCR2523.896 ± 0.500bc 0.933 ± 0.026ab24.829 ± 0.526b
    FCR523.617 ± 0.164cd0.960 ± 0.041a24.578 ± 0.204bc
    FCR1323.411 ± 0.145cd 0.763 ± 0.043de24.174 ± 0.139cd0.267 ± 0.003b 0.729 ± 0.003c2.725 ± 0.048d 0.003 7
    FCR1523.370 ± 0.049cd 0.738 ± 0.096def24.108 ± 0.145cd
    FCR2423.248 ± 0.022de 0.666 ± 0.143ef23.914 ± 0.124d
    FCR123.206 ± 0.432de 0.733 ± 0.089def23.938 ± 0.371cd
    FCR322.708 ± 0.566ef 0.898 ± 0.029abc23.606 ± 0.541de
    FCR3222.385 ± 0.426fg 0.752 ± 0.048de23.137 ± 0.395ef
    WT22.042 ± 0.337g 0.845 ± 0.046abcd22.887 ± 0.331fg0.259 ± 0.002cd0.736 ± 0.002b2.841 ± 0.024bc0.004 3
    FCR222.016 ± 0.356g 0.669 ± 0.058ef22.685 ± 0.380fg0.262 ± 0.001c 0.734 ± 0.001b2.796 ± 0.017c 0.004 4
    FCR3322.010 ± 0.497g 0.674 ± 0.037ef22.683 ± 0.534fg
    FCR2721.772 ± 0.579g 0.611 ± 0.055f 22.383 ± 0.571g
    注:“—” 代表未测定的数据;表中不同字母表示在0.05水平上差异显著;表中数据表示形式为均值 ± 标准差。下同。Notes: “—” stands for unmeasured values. Different letters mean significant difference at P < 0.05 level; data in the table are mean ± standard deviation. The same below.
    下载: 导出CSV

    表  3   BpCCR1株系纤维长、宽及基本密度多重比较

    Table  3   Multiple comparisons of fiber length, width and basic density of BpCCR1 transgenic lines

    基因
    Gene
    株系
    Line
    木纤维 Wood fiber基本密度
    Basic density/(g·cm− 3)
    长 Length/μm宽 Width/μm长/宽 Length/width
    正义链 SenseCR15661.2 ± 55.5b14.4 ± 2.2b46.7 ± 3.8b0.365 5 ± 0.001 1d
    CR13657.5 ± 42.4bc14.9 ± 2.3b45.0 ± 3.8b0.392 2 ± 0.001 5b
    CR4637.0 ± 52.4c14.2 ± 2.1b45.7 ± 3.9b0.382 0 ± 0.003 9c
    CR11652.4 ± 37.9bc14.7 ± 2.5b45.4 ± 4.7b0.382 9 ± 0.008 9c
    WT650.4 ± 38.8bc16.1 ± 1.8a40.8 ± 2.3c0.381 0 ± 0.001 3c
    CR8798.3 ± 48.2a15.2 ± 1.9b53.4 ± 3.9a0.423 5 ± 0.006 7a
    反义链 AntisenseFCR11619.6 ± 38.8cd15.3 ± 1.9def41.2 ± 5.4a0.387 7 ± 0.004 5bcd
    FCR8622.2 ± 37.1cd15.4 ± 1.9def41.0 ± 5.3a0.371 9 ± 0.001 3g
    FCR36605.9 ± 43.9de16.7 ± 2.1bc36.5 ± 3.6b0.382 1 ± 0.007 9def
    FCR25562.3 ± 29.9f15.1 ± 1.5ef37.5 ± 3.9b0.376 4 ± 0.005 2fg
    FCR5587.1 ± 37.0e16.3 ± 2.8bcde37.0 ± 5.9b0.387 4 ± 0.002 1bcd
    FCR13469.6 ± 30.9h13.5 ± 1.9g35.3 ± 4.8bc0.411 0 ± 0.002 6a
    FCR15638.1 ± 31.9bc17.4 ± 2.7b37.3 ± 4.9b0.381 6 ± 0.005 3def
    FCR24610.4 ± 58.4d15.1 ± 2.2f41.1 ± 5.3a0.378 0 ± 0.000 4efg
    FCR1708.6 ± 55.4a17.3 ± 2.6b42.3 ± 4.8a0.373 3 ± 0.003 0fg
    FCR3559.2 ± 51.5f16.0 ± 2.9cdef35.9 ± 6.0bc0.379 1 ± 0.001 9defg
    FCR32544.7 ± 37.6f16.3 ± 2.1bcd33.7 ± 4.4c0.386 0 ± 0.007 9cde
    WT650.4 ± 38.8b16.1 ± 1.9cdef40.8 ± 4.3a0.381 0 ± 0.001 3def
    FCR2640.1 ± 43.9bc15.6 ± 2.9cdef42.6 ± 4.8a0.395 6 ± 0.008 0b
    FCR33609.3 ± 40.5d16.6 ± 2.0bc37.2 ± 4.5b0.385 7 ± 0.000 3cde
    FCR27496.8 ± 37.9g18.5 ± 2.2a27.2 ± 3.6d0.394 0 ± 0.005 7bc
    下载: 导出CSV

    表  4   BpCCR1基因株系树高、地径、胸径及材积多重比较

    Table  4   Multiple comparisons of tree height, ground diameter, DBH and volume of BpCCR1 transgenic lines

    基因 Gene   株系 Line树高 Tree height/m地径 Ground diameter/mm胸径 DBH/mm材积 Volume/cm3
    正义链 SenseCR13421.5 ± 21.5a32.4 ± 1.2ab16.3 ± 1.2b400.0 ± 32.2a
    CR4402.0 ± 11.5ab33.2 ± 1.3a17.7 ± 0.8ab447.3 ± 30.5a
    CR15388.3 ± 26.4abc30.3 ± 0.9ab18.0 ± 0.9ab446.0 ± 39.5a
    WT368.8 ± 24.8bc28.1 ± 1.6b16.7 ± 0.8b374.0 ± 20.8a
    CR11352.0 ± 10.2cd30.1 ± 0.2ab19.6 ± 1.0a473.3 ± 26.8a
    CR8310.5 ± 13.5d28.3 ± 0.9b13.8 ± 0.8c217.6 ± 31.8b
    反义链 AntisenseFCR2467.5 ± 14.5a39.1 ± 1.7a25.1 ± 1.4a993.4 ± 41.4a
    FCR11460.7 ± 16.2ab36.1 ± 2.9abc23.8 ± 0.6a889.3 ± 33.8b
    FCR8440.5 ± 12.5abc38.4 ± 2.5a20.0 ± 1.2b612.6 ± 28.9c
    FCR36432.5 ± 16.5bc31.7 ± 1.7cde17.7 ± 1.0cd483.5 ± 40.8de
    FCR33420.3 ± 6.7cd33.4 ± 0.5bcd19.7 ± 0.5b571.3 ± 20.4cd
    FCR27410.0 ± 10.0cd37.5 ± 2.2ab20.5 ± 1.3b597.4 ± 47.8c
    FCR32396.0 ± 6.0de37.1 ± 0.6ab18.8 ± 0.0bc489.6 ± 4.9de
    FCR5390.7 ± 17.8def29.6 ± 2.5de16.9 ± 0.7cde398.3 ± 27.4ef
    FCR1390.5 ± 12.5def30.2 ± 0.8de16.7 ± 0.6de391.6 ± 21.4ef
    FCR15389.0 ± 0.0def30.5 ± 0.0de17.0 ± 0.0cde400.9 ± 0.0ef
    FCR24373.0 ± 7.0ef34.3 ± 1.0abcd17.8 ± 1.3cd421.6 ± 38.1ef
    WT368.8 ± 24.8ef28.1 ± 1.6ef16.7 ± 0.8de374.0 ± 40.8f
    FCR3356.7 ± 10.8f23.9 ± 1.7f11.9 ± 0.6g191.1 ± 8.6g
    FCR25310.0 ± 5.0g30.4 ± 2.1de15.5 ± 0.5ef268.5 ± 11.8g
    FCR13306.0 ± 36.1g31.5 ± 1.3cde14.3 ± 1.6f233.9 ± 43.2g
    下载: 导出CSV

    表  5   特征根及标准化特征向量

    Table  5   Characteristic roots and standardized eigenvectors

    主成分
    Main component
    特征根
    Characteristic root
    方差贡献率
    Variance contribution rate/%
    累积贡献率
    Cumulative contribution rate/%
    性状
    Trait
    因子载荷1
    Factor loading 1
    特征向量1
    Standardized eigenvector 1
    因子载荷2
    Factor loading 2
    特征向量2
    Standardized eigenvector 2
    Y12.85247.52647.526树高
    Tree height (X1)
    0.9280.549 5− 0.003− 0.002 5
    Y21.40223.37370.900胸径
    DBH (X2)
    0.9270.548 9 0.278 0.234 8
    Y30.92915.48286.382材积
    Volume (X3)
    0.9490.561 9 0.275 0.232 3
    Y40.60210.03896.420总木质素含量
    Total lignin content (X4)
    0.2370.140 3− 0.766− 0.646 9
    Y50.203 3.39199.811纤维长/宽
    Fiber length/
    width (X5)
    − 0.155 − 0.091 8 0.404 0.341 2
    Y60.011 0.189100.000 密度
    Density (X6)
    − 0.386 − 0.228 6 0.706 0.596 3
    下载: 导出CSV

    表  6   参试株系综合评价

    Table  6   Comprehensive evaluation of each tested lines

    株系 
    Line 
    标准分 Standardized valueY1排名
    Rank
    Y2排名
    Rank
    X1X2X3X4X5X6
    WT− 0.442 3− 0.375 1− 0.453 0− 0.889 70.123 7− 0.369 8− 0.755 1160.205 08
    CR40.273 2− 0.064 3− 0.088 2− 0.298 70.982 9− 0.288 1− 0.001 090.320 57
    CR8− 1.698 6− 1.323 3− 1.230 8− 1.636 22.334 02.859 3− 3.448 8202.967 41
    CR11− 0.804 30.553 20.040 8− 0.733 70.933 9− 0.223 6− 0.253 0140.801 34
    CR130.693 4− 0.520 5− 0.323 7− 0.056 60.854 60.482 0− 0.283 1150.416 56
    CR15− 0.021 30.037 8− 0.094 80.095 11.150 5− 1.545 10.216 78− 0.603 413
    FCR10.025 4− 0.372 2− 0.365 30.195 10.381 9− 0.951 9− 0.185 713− 0.735 914
    FCR21.684 72.323 22.627 7− 1.097 70.432 90.735 13.315 712.447 92
    FCR3− 0.703 7− 1.912 8− 1.362 6− 0.147 3− 0.748 8− 0.510 7− 2.037 518− 1.228 618
    FCR50.029 0− 0.326 5− 0.332 00.854 3− 0.559 7− 0.719 1− 0.014 310− 1.326 220
    FCR81.102 80.672 50.734 01.449 50.154 5− 1.054 01.817 73− 1.187 816
    FCR111.537 41.911 12.109 81.897 70.189 50.142 93.295 52− 0.142 811
    FCR13− 1.795 6− 1.153 0− 1.149 70.438 4− 0.861 91.909 3− 2.561 4190.027 610
    FCR15− 0.007 0− 0.286 7− 0.319 30.370 0− 0.497 0− 0.321 9− 0.169 511− 0.742 415
    FCR24− 0.351 8− 0.020 7− 0.216 00.170 40.163 6− 0.596 3− 0.180 812− 0.464 212
    FCR25− 1.709 4− 0.776 8− 0.977 41.113 5− 0.469 80.119 4− 1.742 817− 1.214 617
    FCR270.445 60.822 40.658 3− 1.409 0− 2.277 50.615 70.936 840.846 53
    FCR320.143 90.277 50.122 0− 0.631 6− 1.131 00.010 70.312 770.122 29
    FCR330.668 30.590 30.528 3− 1.099 3− 0.523 0− 0.009 80.884 150.786 55
    FCR360.930 4− 0.056 20.091 91.415 8− 0.633 3− 0.284 00.853 86− 1.295 519
    下载: 导出CSV
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出版历程
  • 收稿日期:  2018-12-17
  • 修回日期:  2019-03-04
  • 网络出版日期:  2019-05-30
  • 发布日期:  2019-05-31

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