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
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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qRTPCR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。
[Objective] Cinnamoyl-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. [Method] 7-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. [Result] PCR 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. [Conclusion] Both 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.
[Objective] Cinnamoyl-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. [Method] 7-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. [Result] PCR 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. [Conclusion] Both 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.
引文
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[2]胡可,严雪锋,栗丹,等.沉默CCR和CAD基因培育低木质素含量转基因多年生黑麦草[J].草业学报,2013,22(5):72-83.Hu K,Yan X F,Li D,et al.Genetic improvement of perennial ryegrass with low lignin content by silencing genes of CCR and CAD[J].Acta Prataculturae Sinica,2013,22(5):72-83.
[3]Chen H C,Song J,Wang J P,et al.Systems biology of lignin biosynthesis in Populus trichocarpa:heteromeric 4-coumaric acid:coenzyme A ligase protein complex formation,regulation,and numerical modeling[J].Plant Cell,2014,26(3):876-893.
[4]高原,陈信波,张志扬.木质素生物合成途径及其基因调控的研究进展[J].生物技术通报,2007(2):47-51.Gao Y,Chen X B,Zhang Z Y.Advances in research on lignin biosynthesis and its molecular regulation[J].Biotechnology Bulletin,2007(2):47-51.
[5]国增超,侯静,郭炜,等.簸箕柳材性性状株内纵向变异的趋势分析[J].南京林业大学学报(自然科学版),2014,38(5):149-152.Guo Z C,Hou J,Guo W,et al.Variation trends of wood property along stem in Salix suchowensis[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2014,38(5):149-152.
[6]Lacombe E,Hawkins S,Van D J,et al.Cinnamoyl CoAreductase,the first committed enzyme of the lignin branch biosynthetic pathway:cloning,expression and phylogenetic relationships[J].The Plant Journal,1997,11(3):429-441.
[7]李波,梁颖,柴友荣.植物肉桂酰辅酶A还原酶(CCR)基因的研究进展[J].分子植物育种,2006,4(增刊1):55-65.Li B,Liang Y,Chai Y R.Achievements in research on plant cinnamoyl-CoA reductase(CCR)genes[J].Molecular Plant Breeding,2006,4(Suppl.1):55-65.
[8]李魏,谭晓风,陈鸿鹏.植物肉桂酰辅酶A还原酶基因的结构功能及应用潜力[J].经济林研究,2009,27(1):7-12.Li W,Tan X F,Chen H P.Structure,function and application potential of cinnamoyl-CoA reductase(CCR)gene in plant[J].Nonwood Forest Research,2009,27(1):7-12.
[9]Wadenback J,Arnold S V,Egertsdotter U,et al.Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase(CCR)[J].Transgenic Research,2008,17(3):379-392.
[10]Rest V D B.Down-regulation of cinnamoyl-CoA reductase in tomato(Solanum lycopersicum L.)induces dramatic changes in soluble phenolic pools[J].Journal of Experimental Botany,2006,57(6):1399-1411.
[11]秋增昌,王海毅.木质素的应用研究现状与进展[J].西南造纸,2004,33(3):29-33.Qiu Z C,Wang H Y.Current status and progress of application research of lignin[J].Southwest Pulp and Paper,2004,33(3):29-33.
[12]刘宇,徐焕文,尚福强,等.16年生白桦种源变异及区划[J].林业科学,2016,52(9):48-56.Liu Y,Xu H W,Shang F Q,et al.Variation and zoning of 16-year-old Betula platyphylla provenance[J].Scientia Silvae Sinicae,2016,52(9):48-56.
[13]韦睿.白桦木质素BpCCR1基因的克隆及遗传转化[D].哈尔滨:东北林业大学,2012.Wei R.Gene clone and genetic transformation of cinnamoyl-CoAreductase gene 1 in Betula platyphylla[D].Harbin:Northeast Forestry University,2012.
[14]王朔,黄海娇,杨光,等.转基因白桦杂种T1代的生长发育及AP1基因的遗传分析[J].北京林业大学学报,2016,38(9):1-7.Wang S,Huang H J,Yang G,et al.Growth and developmental analysis of T1 generation from BpAP1 transgenic birch[J].Journal of Beijing Forestry University,2016,38(9):1-7.
[15]黄海娇,李慧玉,姜静.BpAP1转基因白桦中开花相关基因的时序表达[J].东北林业大学学报,2017,45(1):1-6.Huang H J,Li H Y,Jiang J.Quantitative expression analysis of several flowering-related genes in BpAP1 transgenic birch(Betula platyphylla×Betula pendula)[J].Journal of Northeast Forestry University,2017,45(1):1-6.
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[18]刘超逸,刘桂丰,方功桂,等.四倍体白桦木材纤维性状比较及优良母树选择[J].北京林业大学学报,2017,39(2):9-15.Liu C Y,Liu G F,Fang G G,et al.Comparison of tetraploid Betula platyphylla wood fiber traits and selection of superior seed trees[J].Journal of Beijing Forestry University,2017,39(2):9-15.
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