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    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

    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.

       

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