BpMYB4基因在白桦中的遗传转化及低温胁迫应答反应
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摘要
【目的】为了研究Bp MYB4基因在冷胁迫反应中的功能,对其进行了白桦转化试验。【方法】采用定量PCR技术对Bp MYB4基因在白桦的根、茎、叶、木质部、芽、茎段等18个组织部位的表达模式进行分析,然后克隆构建PGWB2-Bp MYB4植物过表达载体,通过农杆菌介导法进行白桦合子胚的遗传转化,采用PCR和荧光定量PCR技术检测转基因白桦株系中Bp MYB4基因的相对表达量,最后采用分光光度计法对Bp MYB4转基因白桦进行了8项生理指标的测定。【结果】相对于芽的表达量,Bp MYB4基因在第3片叶的叶脉和第1片叶与第2片叶之间嫩茎的表达量最高;白桦的遗传转化总共获得了13个过表达株系,且Bp MYB4基因已经成功整合到白桦基因组上,定量结果发现MYB-8、MYB-11、MYB-12转基因白桦中Bp MYB4基因的表达量相对于非转基因对照株系(NT)上调幅度最大;在低温胁迫下,过量表达Bp MYB4基因提高了白桦的超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、花青素(OPC)含量、过氧化氢酶活性(CAT)、可溶性糖含量、可溶性蛋白含量和脯氨酸(PRO)含量,同时降低了转基因白桦的丙二醛(MDA)的含量。【结论】Bp MYB4转基因白桦在低温胁迫下能够产生具有保护作用的物质,具有一定的抵抗低温的能力,因此,可以将其培育成抗冻优选株系。
【Objective】Our objective was to understand the functions of the Bp MYB4 gene during low-temperature stress.【Method】We performed the following experiments in this study. Firstly,we analyzed the expression pattern of the Bp MYB4 gene in different tissues of birch,including roots,stems,leaves,xylem and buds. Secondly,we constructed the plant overexpression vector of PGWB2-Bp MYB4,and generated birch transgenic lines that overexpressed Bp MYB4 using Agrobacterium-mediated transformation. Then,a polymerase chain reaction( PCR) and quantitative real-time PCR( qRT-PCR) were used to identify the transformants. Finally,we examined eight physiological indexes of transgenic birch under low temperature using spectrophotometry.【Result】Campare with the expression of Bp MYB4 in buds,the highest expression level of Bp MYB4 was in the veins of the third leaf and the tender stems between the first leaf and the second leaf. We obtained 13 independent 35 S ∶ ∶ Bp MYB4 transgenic lines and PCR results provided preliminary evidence that exogenous Bp MYB4 was integrated into the genome of birch. qRT-PCR results indicated that Bp MYB4 expression in MYB-8,MYB-11 and MYB-12 lines was significantly higher than that in the non-transgenic birch( NT). Under cool stress,overexpression of the Bp MYB4 gene in birch increased enzyme activities of SOD,POD and CAT. Overexpression of Bp MYB4 also increased contents of OPC,soluble sugar,soluble protein,and proline. Compared to NT,the content of malondialdehyde( MDA) in transgenic birch was reduced.【Conclusion】The results indicated that Bp MYB4 transgenic birch could produce protective substances under cold stress,and it will have an ability to resist cold. It can be developed into a freeze-resistant preferred strain.
【Objective】Our objective was to understand the functions of the Bp MYB4 gene during low-temperature stress.【Method】We performed the following experiments in this study. Firstly,we analyzed the expression pattern of the Bp MYB4 gene in different tissues of birch,including roots,stems,leaves,xylem and buds. Secondly,we constructed the plant overexpression vector of PGWB2-Bp MYB4,and generated birch transgenic lines that overexpressed Bp MYB4 using Agrobacterium-mediated transformation. Then,a polymerase chain reaction( PCR) and quantitative real-time PCR( qRT-PCR) were used to identify the transformants. Finally,we examined eight physiological indexes of transgenic birch under low temperature using spectrophotometry.【Result】Campare with the expression of Bp MYB4 in buds,the highest expression level of Bp MYB4 was in the veins of the third leaf and the tender stems between the first leaf and the second leaf. We obtained 13 independent 35 S ∶ ∶ Bp MYB4 transgenic lines and PCR results provided preliminary evidence that exogenous Bp MYB4 was integrated into the genome of birch. qRT-PCR results indicated that Bp MYB4 expression in MYB-8,MYB-11 and MYB-12 lines was significantly higher than that in the non-transgenic birch( NT). Under cool stress,overexpression of the Bp MYB4 gene in birch increased enzyme activities of SOD,POD and CAT. Overexpression of Bp MYB4 also increased contents of OPC,soluble sugar,soluble protein,and proline. Compared to NT,the content of malondialdehyde( MDA) in transgenic birch was reduced.【Conclusion】The results indicated that Bp MYB4 transgenic birch could produce protective substances under cold stress,and it will have an ability to resist cold. It can be developed into a freeze-resistant preferred strain.
引文
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[9]SOLTSZ A,VGJFALVI A,RIZZA F,et al. The rice Osmyb4,gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants[J].Journal of Applied Genetics,2012,53(2):133-143. DOI:10.1007/s13353-011-0081-x.
[10]CHANG S,PURYEAR J,CAIRNEY J. A simple and efficient method for isolating RNA from pine trees[J]. Plant Molecular Biology Reporter, 1993, 11(2):113-116. DOI:10.1007IBF02670468.
[11]LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods,2001,25(4):402-408. DOI:10.1006/meth.2001.1262.
[12]董京祥,任丽,张园,等.白桦BpTCPs基因家族生物信息学及时空表达分析[J].南京林业大学学报(自然科学版),2018, 42(4):113-118. DOI:10. 3969/j. issn. 1000-2006.201709001.DONG J X,REN L,ZHANG Y,et al. Bioinformatics and expression analysis of BpTCPs in Betula platyphylla Suk[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2018,42(4):113-118.
[13]周坦洋,罗芙蓉,白彬.葡萄籽原花青素生物药理活性的研究进展[J].哈尔滨医科大学学报,2012,46(1):94-96.DOI:10.3969/j.issn.1000-1905.2012.01.028.ZHOU T Y,LUO F R,BAI B. Advances in the pharmacological activity of proanthocyanidins in grape seed[J]. Journal of Harbin Medical University,2012,46(1):94-96.
[14]赵明明,周余华,彭方仁,等.低温胁迫下冬青叶片细胞内Ca2+水平及可溶性糖含量的变化[J].南京林业大学学报(自然科学版),2013,37(5):1-5. DOI:10.3969/j.issn.1006-2006.2013.05.001.ZHAO M M,ZHOU Y H,PENG F R,et al. Changes of Ca2+level and soluble sugar content in cells of Ilex L. leaflets under low temperature stress[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2013,37(5):1-5.
[15]林善枝,李雪平,张志毅.低温锻炼对毛白杨幼苗抗冻性和总可溶性蛋白质的影响[J].林业科学,2002,38(6):137-141. DOI:10.3321/j.issn:1001-7488.2002.06.022.LIN S Z,LI X P,ZHANG Z Y. The effects of cold acclimation on the freezing resistance and total soluble protein in Populus tomentosa seedlings[J]. Scientia Silvae Sinicae,2002,38(6):137-141.
[16]胡春霞,王丽,汤杰.低温对南果梨的生理生化指标的影响[J].沈阳农业大学学报,2009,40(3):349-352. DOI:10.3969/j.issn.1000-1700.2009.03.021.HU C X,WANG L,TANG J. Effect of low temperature on physiological indicators of Nanguo-Pear[J]. Journal of Shenyang Agricultural University,2009,40(3):349-352.
[17]郭光艳,柏峰,刘伟,等.转录因子对木质素生物合成调控的研究进展[J].中国农业科学,2015,48(7):1277-1287.DOI:10.3864/j.issn.0578-1752.2015.07.03.GUO G Y,BAI F,LIU W,et al. Advances in research of the regulation of transcription factors of lignin biosynthesis[J].Scientia Agricultura Sinica,2015,48(7):1277-1287.
[18]王勤礼,许耀照,闫芳,等.低温弱光盐胁迫对辣椒幼苗生长和生理特性的影响[J].中国农学通报,2015,31(22):111-114.WANG Q L,XU Y Z,YAN F,et al. Effects of low temperature,weak light and salt stress on growth and physiological characteristics of pepper seedling[J]. Chinese Agricultural Science Bulletin,2015,31(22):111-114.
[2]刘建芳,周瑞莲,赵梅.作物抗冻机理及抗冻基因研究进展[J].鲁东大学学报(自然科学版),2010,26(3):258-265.DOI:10.3969/j.issn.1673-8020.2010.03.017.LIU J F,ZHOU R L,ZHAO M. Progress in crops mechanism of freezing tolerance and antifreeze gene[J]. Ludong University Journal(Natural Science Edition),2010,26(3):258-265.
[3]乌凤章.白桦低温胁迫响应与叶绿体RNA结合蛋白的蛋白质组学研究[D].哈尔滨:东北林业大学,2008.WU F Z. Proteomic analysis of low temperature tress responses and chloroplast RNA binding proteins in Betula platyphlla[D].Harbin:Northeast Forestry University,2008.
[4]陈清,汤浩茹,董晓莉,等.植物Myb转录因子的研究进展[J].基因组学与应用生物学,2009,28(2):365-372. DOI:10.3969/gab.028.000365.CHEN Q,TANG H R,DONG X L,et al. Progress in the study of plant Myb transcription factors[J]. Genomics and Applied Biology,2009,28(2):365-372.
[5]杜海.植物MYB转录因子家族的分子进化机制及调控类黄酮生物合成MYB基因的鉴定[D].成都:四川农业大学,2013.DU H. Molecular evolution of MYB transcription factor gene family in plants and characterization of the MYB genes involved in flavonoid biosynthesis[D]. Chengdu:Sichuan Agricultural University,2013.
[6]VANNINI C, LOCATELLI F, BRACALE M, et al.Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants[J].Plant Journal,2004,37(1):115-127. DOI:10. 1046/j. 1365-313X. 2003.01938.x.
[7]VANNINI C,IRITI M,BRACALE M,et al. The ectopic expression of the rice Osmyb4,gene in Arabidopsis increases tolerance to abiotic,environmental and biotic stresses[J]. Physiological&Molecular Plant Pathology,2006,69(1):26-42. DOI:10.1016/j.pmpp.2006.12.005.
[8]PARK M R,YUN K Y,MOHANTY B,et al. Supra-optimal expression of the cold-regulated Os Myb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development[J].Plant Cell&Environment,2010,33(12):2209-2230. DOI:10.1111/j.1365-3040.2010.02221.x.
[9]SOLTSZ A,VGJFALVI A,RIZZA F,et al. The rice Osmyb4,gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants[J].Journal of Applied Genetics,2012,53(2):133-143. DOI:10.1007/s13353-011-0081-x.
[10]CHANG S,PURYEAR J,CAIRNEY J. A simple and efficient method for isolating RNA from pine trees[J]. Plant Molecular Biology Reporter, 1993, 11(2):113-116. DOI:10.1007IBF02670468.
[11]LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods,2001,25(4):402-408. DOI:10.1006/meth.2001.1262.
[12]董京祥,任丽,张园,等.白桦BpTCPs基因家族生物信息学及时空表达分析[J].南京林业大学学报(自然科学版),2018, 42(4):113-118. DOI:10. 3969/j. issn. 1000-2006.201709001.DONG J X,REN L,ZHANG Y,et al. Bioinformatics and expression analysis of BpTCPs in Betula platyphylla Suk[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2018,42(4):113-118.
[13]周坦洋,罗芙蓉,白彬.葡萄籽原花青素生物药理活性的研究进展[J].哈尔滨医科大学学报,2012,46(1):94-96.DOI:10.3969/j.issn.1000-1905.2012.01.028.ZHOU T Y,LUO F R,BAI B. Advances in the pharmacological activity of proanthocyanidins in grape seed[J]. Journal of Harbin Medical University,2012,46(1):94-96.
[14]赵明明,周余华,彭方仁,等.低温胁迫下冬青叶片细胞内Ca2+水平及可溶性糖含量的变化[J].南京林业大学学报(自然科学版),2013,37(5):1-5. DOI:10.3969/j.issn.1006-2006.2013.05.001.ZHAO M M,ZHOU Y H,PENG F R,et al. Changes of Ca2+level and soluble sugar content in cells of Ilex L. leaflets under low temperature stress[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2013,37(5):1-5.
[15]林善枝,李雪平,张志毅.低温锻炼对毛白杨幼苗抗冻性和总可溶性蛋白质的影响[J].林业科学,2002,38(6):137-141. DOI:10.3321/j.issn:1001-7488.2002.06.022.LIN S Z,LI X P,ZHANG Z Y. The effects of cold acclimation on the freezing resistance and total soluble protein in Populus tomentosa seedlings[J]. Scientia Silvae Sinicae,2002,38(6):137-141.
[16]胡春霞,王丽,汤杰.低温对南果梨的生理生化指标的影响[J].沈阳农业大学学报,2009,40(3):349-352. DOI:10.3969/j.issn.1000-1700.2009.03.021.HU C X,WANG L,TANG J. Effect of low temperature on physiological indicators of Nanguo-Pear[J]. Journal of Shenyang Agricultural University,2009,40(3):349-352.
[17]郭光艳,柏峰,刘伟,等.转录因子对木质素生物合成调控的研究进展[J].中国农业科学,2015,48(7):1277-1287.DOI:10.3864/j.issn.0578-1752.2015.07.03.GUO G Y,BAI F,LIU W,et al. Advances in research of the regulation of transcription factors of lignin biosynthesis[J].Scientia Agricultura Sinica,2015,48(7):1277-1287.
[18]王勤礼,许耀照,闫芳,等.低温弱光盐胁迫对辣椒幼苗生长和生理特性的影响[J].中国农学通报,2015,31(22):111-114.WANG Q L,XU Y Z,YAN F,et al. Effects of low temperature,weak light and salt stress on growth and physiological characteristics of pepper seedling[J]. Chinese Agricultural Science Bulletin,2015,31(22):111-114.