Effects of transgenic sense and antisense of BpCCR1 on 7-year-old potted birch and selection of excellent lines
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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。结论转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.
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Keywords:
- Betula platyphylla /
- CCR /
- lignin /
- fiber /
- excellent line
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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[2−4]。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(Eucalyptus)CCR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。
1. 材料与方法
1.1 材 料
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。
1.2 方 法
1.2.1 转基因株系的分子检测
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 rRNA GAGGTAGCTTCGGGCGCAACT GCAGGTTAGCGAAATGCGATAC BpCCR1 AGCATGTGCGAGAACACCATC ACTCATCACTCCAGCAGCCA 内源BpCCR1 Endogenous BpCCR1 CAAGAATGCAGCAGGCAGATAC GAGAGAGGTGACATAAACGGCC 1.2.2 材性性状测定
(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[16−17]。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]。
1.2.3 生长量的测定
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[21−22],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。
1.2.4 数据处理
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。
2. 结果与分析
2.1 BpCCR1在白桦转基因株系中稳定表达
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 2 转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-PCR2.2 白桦转基因株系木质素及木质素单体含量分析
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 转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 ligninG S S/G H 正义链
SenseCR15 23.098 ± 0.206a 0.744 ± 0.015a 23.841 ± 0.192a 0.261 ± 0.002cd 0.726 ± 0.002c 2.779 ± 0.035cd 0.012 8 CR13 22.827 ± 0.287ab 0.867 ± 0.158a 23.694 ± 0.160ab 0.280 ± 0.006a 0.715 ± 0.006d 2.552 ± 0.070e 0.004 6 CR4 22.683 ± 0.392ab 0.776 ± 0.012a 23.459 ± 0.399abc — — — — CR11 22.472 ± 0.497ab 0.566 ± 0.035b 23.038 ± 0.506bc — — — — WT 22.042 ± 0.337b 0.845 ± 0.046a 22.887 ± 0.331c 0.259 ± 0.002cd 0.736 ± 0.002b 2.841 ± 0.024bc 0.004 3 CR8 21.284 ± 0.648c 0.879 ± 0.025a 22.163 ± 0.626d 0.243 ± 0.002e 0.752 ± 0.002a 3.101 ± 0.026a 0.005 0 反义链
AntisenseFCR11 24.745 ± 0.065a 0.844 ± 0.061abcd 25.589 ± 0.091a 0.257 ± 0.001d 0.738 ± 0.001b 2.873 ± 0.011b 0.005 0 FCR8 24.338 ± 0.109ab 0.816 ± 0.111bcd 25.154 ± 0.190ab — — — — FCR36 24.323 ± 0.116ab 0.799 ± 0.061cde 25.122 ± 0.128ab — — — — FCR25 23.896 ± 0.500bc 0.933 ± 0.026ab 24.829 ± 0.526b — — — — FCR5 23.617 ± 0.164cd 0.960 ± 0.041a 24.578 ± 0.204bc — — — — FCR13 23.411 ± 0.145cd 0.763 ± 0.043de 24.174 ± 0.139cd 0.267 ± 0.003b 0.729 ± 0.003c 2.725 ± 0.048d 0.003 7 FCR15 23.370 ± 0.049cd 0.738 ± 0.096def 24.108 ± 0.145cd — — — — FCR24 23.248 ± 0.022de 0.666 ± 0.143ef 23.914 ± 0.124d — — — — FCR1 23.206 ± 0.432de 0.733 ± 0.089def 23.938 ± 0.371cd — — — — FCR3 22.708 ± 0.566ef 0.898 ± 0.029abc 23.606 ± 0.541de — — — — FCR32 22.385 ± 0.426fg 0.752 ± 0.048de 23.137 ± 0.395ef — — — — WT 22.042 ± 0.337g 0.845 ± 0.046abcd 22.887 ± 0.331fg 0.259 ± 0.002cd 0.736 ± 0.002b 2.841 ± 0.024bc 0.004 3 FCR2 22.016 ± 0.356g 0.669 ± 0.058ef 22.685 ± 0.380fg 0.262 ± 0.001c 0.734 ± 0.001b 2.796 ± 0.017c 0.004 4 FCR33 22.010 ± 0.497g 0.674 ± 0.037ef 22.683 ± 0.534fg — — — — FCR27 21.772 ± 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. 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。
2.3 白桦转基因株系木纤维的长、宽以及长/宽的变化
转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 转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 Sense CR15 661.2 ± 55.5b 14.4 ± 2.2b 46.7 ± 3.8b 0.365 5 ± 0.001 1d CR13 657.5 ± 42.4bc 14.9 ± 2.3b 45.0 ± 3.8b 0.392 2 ± 0.001 5b CR4 637.0 ± 52.4c 14.2 ± 2.1b 45.7 ± 3.9b 0.382 0 ± 0.003 9c CR11 652.4 ± 37.9bc 14.7 ± 2.5b 45.4 ± 4.7b 0.382 9 ± 0.008 9c WT 650.4 ± 38.8bc 16.1 ± 1.8a 40.8 ± 2.3c 0.381 0 ± 0.001 3c CR8 798.3 ± 48.2a 15.2 ± 1.9b 53.4 ± 3.9a 0.423 5 ± 0.006 7a 反义链 Antisense FCR11 619.6 ± 38.8cd 15.3 ± 1.9def 41.2 ± 5.4a 0.387 7 ± 0.004 5bcd FCR8 622.2 ± 37.1cd 15.4 ± 1.9def 41.0 ± 5.3a 0.371 9 ± 0.001 3g FCR36 605.9 ± 43.9de 16.7 ± 2.1bc 36.5 ± 3.6b 0.382 1 ± 0.007 9def FCR25 562.3 ± 29.9f 15.1 ± 1.5ef 37.5 ± 3.9b 0.376 4 ± 0.005 2fg FCR5 587.1 ± 37.0e 16.3 ± 2.8bcde 37.0 ± 5.9b 0.387 4 ± 0.002 1bcd FCR13 469.6 ± 30.9h 13.5 ± 1.9g 35.3 ± 4.8bc 0.411 0 ± 0.002 6a FCR15 638.1 ± 31.9bc 17.4 ± 2.7b 37.3 ± 4.9b 0.381 6 ± 0.005 3def FCR24 610.4 ± 58.4d 15.1 ± 2.2f 41.1 ± 5.3a 0.378 0 ± 0.000 4efg FCR1 708.6 ± 55.4a 17.3 ± 2.6b 42.3 ± 4.8a 0.373 3 ± 0.003 0fg FCR3 559.2 ± 51.5f 16.0 ± 2.9cdef 35.9 ± 6.0bc 0.379 1 ± 0.001 9defg FCR32 544.7 ± 37.6f 16.3 ± 2.1bcd 33.7 ± 4.4c 0.386 0 ± 0.007 9cde WT 650.4 ± 38.8b 16.1 ± 1.9cdef 40.8 ± 4.3a 0.381 0 ± 0.001 3def FCR2 640.1 ± 43.9bc 15.6 ± 2.9cdef 42.6 ± 4.8a 0.395 6 ± 0.008 0b FCR33 609.3 ± 40.5d 16.6 ± 2.0bc 37.2 ± 4.5b 0.385 7 ± 0.000 3cde FCR27 496.8 ± 37.9g 18.5 ± 2.2a 27.2 ± 3.6d 0.394 0 ± 0.005 7bc 2.4 白桦转基因株系的木材基本密度比较
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。
2.5 白桦转基因株系的生长性状分析
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 转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 Sense CR13 421.5 ± 21.5a 32.4 ± 1.2ab 16.3 ± 1.2b 400.0 ± 32.2a CR4 402.0 ± 11.5ab 33.2 ± 1.3a 17.7 ± 0.8ab 447.3 ± 30.5a CR15 388.3 ± 26.4abc 30.3 ± 0.9ab 18.0 ± 0.9ab 446.0 ± 39.5a WT 368.8 ± 24.8bc 28.1 ± 1.6b 16.7 ± 0.8b 374.0 ± 20.8a CR11 352.0 ± 10.2cd 30.1 ± 0.2ab 19.6 ± 1.0a 473.3 ± 26.8a CR8 310.5 ± 13.5d 28.3 ± 0.9b 13.8 ± 0.8c 217.6 ± 31.8b 反义链 Antisense FCR2 467.5 ± 14.5a 39.1 ± 1.7a 25.1 ± 1.4a 993.4 ± 41.4a FCR11 460.7 ± 16.2ab 36.1 ± 2.9abc 23.8 ± 0.6a 889.3 ± 33.8b FCR8 440.5 ± 12.5abc 38.4 ± 2.5a 20.0 ± 1.2b 612.6 ± 28.9c FCR36 432.5 ± 16.5bc 31.7 ± 1.7cde 17.7 ± 1.0cd 483.5 ± 40.8de FCR33 420.3 ± 6.7cd 33.4 ± 0.5bcd 19.7 ± 0.5b 571.3 ± 20.4cd FCR27 410.0 ± 10.0cd 37.5 ± 2.2ab 20.5 ± 1.3b 597.4 ± 47.8c FCR32 396.0 ± 6.0de 37.1 ± 0.6ab 18.8 ± 0.0bc 489.6 ± 4.9de FCR5 390.7 ± 17.8def 29.6 ± 2.5de 16.9 ± 0.7cde 398.3 ± 27.4ef FCR1 390.5 ± 12.5def 30.2 ± 0.8de 16.7 ± 0.6de 391.6 ± 21.4ef FCR15 389.0 ± 0.0def 30.5 ± 0.0de 17.0 ± 0.0cde 400.9 ± 0.0ef FCR24 373.0 ± 7.0ef 34.3 ± 1.0abcd 17.8 ± 1.3cd 421.6 ± 38.1ef WT 368.8 ± 24.8ef 28.1 ± 1.6ef 16.7 ± 0.8de 374.0 ± 40.8f FCR3 356.7 ± 10.8f 23.9 ± 1.7f 11.9 ± 0.6g 191.1 ± 8.6g FCR25 310.0 ± 5.0g 30.4 ± 2.1de 15.5 ± 0.5ef 268.5 ± 11.8g FCR13 306.0 ± 36.1g 31.5 ± 1.3cde 14.3 ± 1.6f 233.9 ± 43.2g 2.6 白桦转基因株系的综合评价
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 2Y1 2.852 47.526 47.526 树高
Tree height (X1)0.928 0.549 5 − 0.003 − 0.002 5 Y2 1.402 23.373 70.900 胸径
DBH (X2)0.927 0.548 9 0.278 0.234 8 Y3 0.929 15.482 86.382 材积
Volume (X3)0.949 0.561 9 0.275 0.232 3 Y4 0.602 10.038 96.420 总木质素含量
Total lignin content (X4)0.237 0.140 3 − 0.766 − 0.646 9 Y5 0.203 3.391 99.811 纤维长/宽
Fiber length/
width (X5)− 0.155 − 0.091 8 0.404 0.341 2 Y6 0.011 0.189 100.000 密度
Density (X6)− 0.386 − 0.228 6 0.706 0.596 3 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 value Y1 排名
RankY2 排名
RankX1 X2 X3 X4 X5 X6 WT − 0.442 3 − 0.375 1 − 0.453 0 − 0.889 7 0.123 7 − 0.369 8 − 0.755 1 16 0.205 0 8 CR4 0.273 2 − 0.064 3 − 0.088 2 − 0.298 7 0.982 9 − 0.288 1 − 0.001 0 9 0.320 5 7 CR8 − 1.698 6 − 1.323 3 − 1.230 8 − 1.636 2 2.334 0 2.859 3 − 3.448 8 20 2.967 4 1 CR11 − 0.804 3 0.553 2 0.040 8 − 0.733 7 0.933 9 − 0.223 6 − 0.253 0 14 0.801 3 4 CR13 0.693 4 − 0.520 5 − 0.323 7 − 0.056 6 0.854 6 0.482 0 − 0.283 1 15 0.416 5 6 CR15 − 0.021 3 0.037 8 − 0.094 8 0.095 1 1.150 5 − 1.545 1 0.216 7 8 − 0.603 4 13 FCR1 0.025 4 − 0.372 2 − 0.365 3 0.195 1 0.381 9 − 0.951 9 − 0.185 7 13 − 0.735 9 14 FCR2 1.684 7 2.323 2 2.627 7 − 1.097 7 0.432 9 0.735 1 3.315 7 1 2.447 9 2 FCR3 − 0.703 7 − 1.912 8 − 1.362 6 − 0.147 3 − 0.748 8 − 0.510 7 − 2.037 5 18 − 1.228 6 18 FCR5 0.029 0 − 0.326 5 − 0.332 0 0.854 3 − 0.559 7 − 0.719 1 − 0.014 3 10 − 1.326 2 20 FCR8 1.102 8 0.672 5 0.734 0 1.449 5 0.154 5 − 1.054 0 1.817 7 3 − 1.187 8 16 FCR11 1.537 4 1.911 1 2.109 8 1.897 7 0.189 5 0.142 9 3.295 5 2 − 0.142 8 11 FCR13 − 1.795 6 − 1.153 0 − 1.149 7 0.438 4 − 0.861 9 1.909 3 − 2.561 4 19 0.027 6 10 FCR15 − 0.007 0 − 0.286 7 − 0.319 3 0.370 0 − 0.497 0 − 0.321 9 − 0.169 5 11 − 0.742 4 15 FCR24 − 0.351 8 − 0.020 7 − 0.216 0 0.170 4 0.163 6 − 0.596 3 − 0.180 8 12 − 0.464 2 12 FCR25 − 1.709 4 − 0.776 8 − 0.977 4 1.113 5 − 0.469 8 0.119 4 − 1.742 8 17 − 1.214 6 17 FCR27 0.445 6 0.822 4 0.658 3 − 1.409 0 − 2.277 5 0.615 7 0.936 8 4 0.846 5 3 FCR32 0.143 9 0.277 5 0.122 0 − 0.631 6 − 1.131 0 0.010 7 0.312 7 7 0.122 2 9 FCR33 0.668 3 0.590 3 0.528 3 − 1.099 3 − 0.523 0 − 0.009 8 0.884 1 5 0.786 5 5 FCR36 0.930 4 − 0.056 2 0.091 9 1.415 8 − 0.633 3 − 0.284 0 0.853 8 6 − 1.295 5 19 Y1=0.5495X1+0.5489X2+0.5619X3+0.1403X4−0.0918X5−0.2286X6 (1) Y2=−0.0025X1+0.2348X2+0.2323X3−0.6469X4+0.3412X5+0.5963X6 (2) 式中:X1、X2、X3、X4、X5、X6分别表示树高、胸径、材积、总木质素含量、纤维长/宽、密度;Y1、Y2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Y1和Y2值进行选择,FCR2、FCR27和FCR33株系为优良株系。
3. 讨 论
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 aestivum)TaCCR1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[26−27]。
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[31−32]。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。
4. 结 论
转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。
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图 2 转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
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 rRNA GAGGTAGCTTCGGGCGCAACT GCAGGTTAGCGAAATGCGATAC BpCCR1 AGCATGTGCGAGAACACCATC ACTCATCACTCCAGCAGCCA 内源BpCCR1 Endogenous BpCCR1 CAAGAATGCAGCAGGCAGATAC GAGAGAGGTGACATAAACGGCC 表 2 转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 ligninG S S/G H 正义链
SenseCR15 23.098 ± 0.206a 0.744 ± 0.015a 23.841 ± 0.192a 0.261 ± 0.002cd 0.726 ± 0.002c 2.779 ± 0.035cd 0.012 8 CR13 22.827 ± 0.287ab 0.867 ± 0.158a 23.694 ± 0.160ab 0.280 ± 0.006a 0.715 ± 0.006d 2.552 ± 0.070e 0.004 6 CR4 22.683 ± 0.392ab 0.776 ± 0.012a 23.459 ± 0.399abc — — — — CR11 22.472 ± 0.497ab 0.566 ± 0.035b 23.038 ± 0.506bc — — — — WT 22.042 ± 0.337b 0.845 ± 0.046a 22.887 ± 0.331c 0.259 ± 0.002cd 0.736 ± 0.002b 2.841 ± 0.024bc 0.004 3 CR8 21.284 ± 0.648c 0.879 ± 0.025a 22.163 ± 0.626d 0.243 ± 0.002e 0.752 ± 0.002a 3.101 ± 0.026a 0.005 0 反义链
AntisenseFCR11 24.745 ± 0.065a 0.844 ± 0.061abcd 25.589 ± 0.091a 0.257 ± 0.001d 0.738 ± 0.001b 2.873 ± 0.011b 0.005 0 FCR8 24.338 ± 0.109ab 0.816 ± 0.111bcd 25.154 ± 0.190ab — — — — FCR36 24.323 ± 0.116ab 0.799 ± 0.061cde 25.122 ± 0.128ab — — — — FCR25 23.896 ± 0.500bc 0.933 ± 0.026ab 24.829 ± 0.526b — — — — FCR5 23.617 ± 0.164cd 0.960 ± 0.041a 24.578 ± 0.204bc — — — — FCR13 23.411 ± 0.145cd 0.763 ± 0.043de 24.174 ± 0.139cd 0.267 ± 0.003b 0.729 ± 0.003c 2.725 ± 0.048d 0.003 7 FCR15 23.370 ± 0.049cd 0.738 ± 0.096def 24.108 ± 0.145cd — — — — FCR24 23.248 ± 0.022de 0.666 ± 0.143ef 23.914 ± 0.124d — — — — FCR1 23.206 ± 0.432de 0.733 ± 0.089def 23.938 ± 0.371cd — — — — FCR3 22.708 ± 0.566ef 0.898 ± 0.029abc 23.606 ± 0.541de — — — — FCR32 22.385 ± 0.426fg 0.752 ± 0.048de 23.137 ± 0.395ef — — — — WT 22.042 ± 0.337g 0.845 ± 0.046abcd 22.887 ± 0.331fg 0.259 ± 0.002cd 0.736 ± 0.002b 2.841 ± 0.024bc 0.004 3 FCR2 22.016 ± 0.356g 0.669 ± 0.058ef 22.685 ± 0.380fg 0.262 ± 0.001c 0.734 ± 0.001b 2.796 ± 0.017c 0.004 4 FCR33 22.010 ± 0.497g 0.674 ± 0.037ef 22.683 ± 0.534fg — — — — FCR27 21.772 ± 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. 表 3 转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 Sense CR15 661.2 ± 55.5b 14.4 ± 2.2b 46.7 ± 3.8b 0.365 5 ± 0.001 1d CR13 657.5 ± 42.4bc 14.9 ± 2.3b 45.0 ± 3.8b 0.392 2 ± 0.001 5b CR4 637.0 ± 52.4c 14.2 ± 2.1b 45.7 ± 3.9b 0.382 0 ± 0.003 9c CR11 652.4 ± 37.9bc 14.7 ± 2.5b 45.4 ± 4.7b 0.382 9 ± 0.008 9c WT 650.4 ± 38.8bc 16.1 ± 1.8a 40.8 ± 2.3c 0.381 0 ± 0.001 3c CR8 798.3 ± 48.2a 15.2 ± 1.9b 53.4 ± 3.9a 0.423 5 ± 0.006 7a 反义链 Antisense FCR11 619.6 ± 38.8cd 15.3 ± 1.9def 41.2 ± 5.4a 0.387 7 ± 0.004 5bcd FCR8 622.2 ± 37.1cd 15.4 ± 1.9def 41.0 ± 5.3a 0.371 9 ± 0.001 3g FCR36 605.9 ± 43.9de 16.7 ± 2.1bc 36.5 ± 3.6b 0.382 1 ± 0.007 9def FCR25 562.3 ± 29.9f 15.1 ± 1.5ef 37.5 ± 3.9b 0.376 4 ± 0.005 2fg FCR5 587.1 ± 37.0e 16.3 ± 2.8bcde 37.0 ± 5.9b 0.387 4 ± 0.002 1bcd FCR13 469.6 ± 30.9h 13.5 ± 1.9g 35.3 ± 4.8bc 0.411 0 ± 0.002 6a FCR15 638.1 ± 31.9bc 17.4 ± 2.7b 37.3 ± 4.9b 0.381 6 ± 0.005 3def FCR24 610.4 ± 58.4d 15.1 ± 2.2f 41.1 ± 5.3a 0.378 0 ± 0.000 4efg FCR1 708.6 ± 55.4a 17.3 ± 2.6b 42.3 ± 4.8a 0.373 3 ± 0.003 0fg FCR3 559.2 ± 51.5f 16.0 ± 2.9cdef 35.9 ± 6.0bc 0.379 1 ± 0.001 9defg FCR32 544.7 ± 37.6f 16.3 ± 2.1bcd 33.7 ± 4.4c 0.386 0 ± 0.007 9cde WT 650.4 ± 38.8b 16.1 ± 1.9cdef 40.8 ± 4.3a 0.381 0 ± 0.001 3def FCR2 640.1 ± 43.9bc 15.6 ± 2.9cdef 42.6 ± 4.8a 0.395 6 ± 0.008 0b FCR33 609.3 ± 40.5d 16.6 ± 2.0bc 37.2 ± 4.5b 0.385 7 ± 0.000 3cde FCR27 496.8 ± 37.9g 18.5 ± 2.2a 27.2 ± 3.6d 0.394 0 ± 0.005 7bc 表 4 转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 Sense CR13 421.5 ± 21.5a 32.4 ± 1.2ab 16.3 ± 1.2b 400.0 ± 32.2a CR4 402.0 ± 11.5ab 33.2 ± 1.3a 17.7 ± 0.8ab 447.3 ± 30.5a CR15 388.3 ± 26.4abc 30.3 ± 0.9ab 18.0 ± 0.9ab 446.0 ± 39.5a WT 368.8 ± 24.8bc 28.1 ± 1.6b 16.7 ± 0.8b 374.0 ± 20.8a CR11 352.0 ± 10.2cd 30.1 ± 0.2ab 19.6 ± 1.0a 473.3 ± 26.8a CR8 310.5 ± 13.5d 28.3 ± 0.9b 13.8 ± 0.8c 217.6 ± 31.8b 反义链 Antisense FCR2 467.5 ± 14.5a 39.1 ± 1.7a 25.1 ± 1.4a 993.4 ± 41.4a FCR11 460.7 ± 16.2ab 36.1 ± 2.9abc 23.8 ± 0.6a 889.3 ± 33.8b FCR8 440.5 ± 12.5abc 38.4 ± 2.5a 20.0 ± 1.2b 612.6 ± 28.9c FCR36 432.5 ± 16.5bc 31.7 ± 1.7cde 17.7 ± 1.0cd 483.5 ± 40.8de FCR33 420.3 ± 6.7cd 33.4 ± 0.5bcd 19.7 ± 0.5b 571.3 ± 20.4cd FCR27 410.0 ± 10.0cd 37.5 ± 2.2ab 20.5 ± 1.3b 597.4 ± 47.8c FCR32 396.0 ± 6.0de 37.1 ± 0.6ab 18.8 ± 0.0bc 489.6 ± 4.9de FCR5 390.7 ± 17.8def 29.6 ± 2.5de 16.9 ± 0.7cde 398.3 ± 27.4ef FCR1 390.5 ± 12.5def 30.2 ± 0.8de 16.7 ± 0.6de 391.6 ± 21.4ef FCR15 389.0 ± 0.0def 30.5 ± 0.0de 17.0 ± 0.0cde 400.9 ± 0.0ef FCR24 373.0 ± 7.0ef 34.3 ± 1.0abcd 17.8 ± 1.3cd 421.6 ± 38.1ef WT 368.8 ± 24.8ef 28.1 ± 1.6ef 16.7 ± 0.8de 374.0 ± 40.8f FCR3 356.7 ± 10.8f 23.9 ± 1.7f 11.9 ± 0.6g 191.1 ± 8.6g FCR25 310.0 ± 5.0g 30.4 ± 2.1de 15.5 ± 0.5ef 268.5 ± 11.8g FCR13 306.0 ± 36.1g 31.5 ± 1.3cde 14.3 ± 1.6f 233.9 ± 43.2g 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 2Y1 2.852 47.526 47.526 树高
Tree height (X1)0.928 0.549 5 − 0.003 − 0.002 5 Y2 1.402 23.373 70.900 胸径
DBH (X2)0.927 0.548 9 0.278 0.234 8 Y3 0.929 15.482 86.382 材积
Volume (X3)0.949 0.561 9 0.275 0.232 3 Y4 0.602 10.038 96.420 总木质素含量
Total lignin content (X4)0.237 0.140 3 − 0.766 − 0.646 9 Y5 0.203 3.391 99.811 纤维长/宽
Fiber length/
width (X5)− 0.155 − 0.091 8 0.404 0.341 2 Y6 0.011 0.189 100.000 密度
Density (X6)− 0.386 − 0.228 6 0.706 0.596 3 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 value Y1 排名
RankY2 排名
RankX1 X2 X3 X4 X5 X6 WT − 0.442 3 − 0.375 1 − 0.453 0 − 0.889 7 0.123 7 − 0.369 8 − 0.755 1 16 0.205 0 8 CR4 0.273 2 − 0.064 3 − 0.088 2 − 0.298 7 0.982 9 − 0.288 1 − 0.001 0 9 0.320 5 7 CR8 − 1.698 6 − 1.323 3 − 1.230 8 − 1.636 2 2.334 0 2.859 3 − 3.448 8 20 2.967 4 1 CR11 − 0.804 3 0.553 2 0.040 8 − 0.733 7 0.933 9 − 0.223 6 − 0.253 0 14 0.801 3 4 CR13 0.693 4 − 0.520 5 − 0.323 7 − 0.056 6 0.854 6 0.482 0 − 0.283 1 15 0.416 5 6 CR15 − 0.021 3 0.037 8 − 0.094 8 0.095 1 1.150 5 − 1.545 1 0.216 7 8 − 0.603 4 13 FCR1 0.025 4 − 0.372 2 − 0.365 3 0.195 1 0.381 9 − 0.951 9 − 0.185 7 13 − 0.735 9 14 FCR2 1.684 7 2.323 2 2.627 7 − 1.097 7 0.432 9 0.735 1 3.315 7 1 2.447 9 2 FCR3 − 0.703 7 − 1.912 8 − 1.362 6 − 0.147 3 − 0.748 8 − 0.510 7 − 2.037 5 18 − 1.228 6 18 FCR5 0.029 0 − 0.326 5 − 0.332 0 0.854 3 − 0.559 7 − 0.719 1 − 0.014 3 10 − 1.326 2 20 FCR8 1.102 8 0.672 5 0.734 0 1.449 5 0.154 5 − 1.054 0 1.817 7 3 − 1.187 8 16 FCR11 1.537 4 1.911 1 2.109 8 1.897 7 0.189 5 0.142 9 3.295 5 2 − 0.142 8 11 FCR13 − 1.795 6 − 1.153 0 − 1.149 7 0.438 4 − 0.861 9 1.909 3 − 2.561 4 19 0.027 6 10 FCR15 − 0.007 0 − 0.286 7 − 0.319 3 0.370 0 − 0.497 0 − 0.321 9 − 0.169 5 11 − 0.742 4 15 FCR24 − 0.351 8 − 0.020 7 − 0.216 0 0.170 4 0.163 6 − 0.596 3 − 0.180 8 12 − 0.464 2 12 FCR25 − 1.709 4 − 0.776 8 − 0.977 4 1.113 5 − 0.469 8 0.119 4 − 1.742 8 17 − 1.214 6 17 FCR27 0.445 6 0.822 4 0.658 3 − 1.409 0 − 2.277 5 0.615 7 0.936 8 4 0.846 5 3 FCR32 0.143 9 0.277 5 0.122 0 − 0.631 6 − 1.131 0 0.010 7 0.312 7 7 0.122 2 9 FCR33 0.668 3 0.590 3 0.528 3 − 1.099 3 − 0.523 0 − 0.009 8 0.884 1 5 0.786 5 5 FCR36 0.930 4 − 0.056 2 0.091 9 1.415 8 − 0.633 3 − 0.284 0 0.853 8 6 − 1.295 5 19 -
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