基于桑树cDNA文库的3个功能基因的诱导表达研究

【作者】 扈东青；

【导师】 赵卫国；

【作者基本信息】 江苏科技大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 桑树是一种重要的经济作物,我国拥有世界上最为丰富的桑树种质资源。但是各种逆境条件和病虫害往往会对桑树的产量造成重大影响,研究桑树一些重要的功能基因及其在胁迫条件下的分子适应机制,可以增强桑树对各种胁迫条件的抗逆性,对桑树种质资源的保存和提高桑树产量有重要作用。与其他作物相比,我们对桑树功能基因的研究至今还处于起步阶段,对桑树的一些功能基因及其编码的蛋白质的功能仍然不甚了解。因此,开展桑树功能基因的研究是一项紧迫而有意义的工作。本文在构建的桑树幼叶cDNA文库的基础上获得了3个ESTs,克隆了这3个基因,并对获得的序列进行了分析和功能推断,用胁迫诱导的方法对这3个基因进行功能验证。本论文的主要研究内容如下:1、桑树几丁质酶基因M-chitinase的克隆及胁迫诱导表达分析从已构建的桑树幼叶cDNA文库得到了一个编码桑树几丁质酶(Chitinase)基因的一段序列,通过RACE与RT-PCR结合获得了这个基因的完整序列,命名为M-chitinase(GeneBank登录号:HQ117891)。序列分析表明,M-chitinase全长1392bp,存在60bp的5’端非翻译序列(5’-UTR)和255bp的3’端非翻译序列(3’-UTR),其开放读码框(ORF)长1077bp,编码358个氨基酸,预测蛋白质分子质量为38.52KD,等电点为4.466。同源分析表明,M-chitinase基因在桑树与卡西猪笼草、玉米、二倍体多年生大刍草等各物种间具有很高的保守性。基于桑树和其它19个物种M-chitinase基因的系统进化分析表明,桑树与野生烟草、湖南烤烟、辣椒、南芥的亲缘关系较近。半定量RT-PCR检测表明,幼叶表达量最高。与正常生长环境相比,M-chitinase基因mRNA的转录水平在NaCl、水杨酸、脱落酸条件下均有明显变化。2、桑树桑树多聚半乳糖醛酸酶抑制蛋白基因M-PGIP的克隆,序列分析及诱导表达研究从已构建的桑树幼叶cDNA文库得到了一个编码桑树多聚半乳糖醛酸酶抑制蛋白(po1yga1acturonase inhibiting protein,PGIP)基因的一段序列,通过RACE与RT-PCR结合首次获得了这个基因的完整序列,命名为M-PGIP(GeneBank登录号:HM044383)。序列分析表明,M-PGIP全长1274bp,存在93bp的5’端非翻译序列(5’-UTR)和179bp的3’端非翻译序列(3’-UTR),其开放读码框(ORF)长1002bp,编码333个氨基酸,预测蛋白质分子质量为37.29KD,等电点为7.25。同源分析表明,M-PGIP基因在桑树与美洲李、桃花、梅花等各物种间具有很高的保守性。基于桑树和其它19个物种M-PGIP基因的系统进化分析表明,桑树与甜瓜、葡萄、葡萄根、猕猴桃的亲缘关系较近。半定量RT-PCR检测表明,幼叶表达量最高。与正常生长环境相比,M-PGIP基因mRNA的转录水平在水杨酸、脱落酸、盐胁迫条件下均有明显变化。并对所克隆的基因进行了原核表达,为今后的转基因植物抗病工程方面打下了良好的基础。3.桑树光合系统IIMpsbR基因的克隆,序列分析及诱导表达研究根据桑树表达序列标签(expressed sequence tags, ESTs),采取RT-PCR方法克隆了一个编码桑树光合系统II PsbR基因的全长cDNA序列,命名为MpsbR(Genbank登录号:GU937873)。序列分析表明,该基因全长623bp,存在56bp的5’-UTR和306bp的3’-UTR,其开放阅读框(ORF)长261bp,编码86个氨基酸,预测蛋白质分子质量为8.95 kD,等电点为11.09。同源分析表明,桑树光合系统II MpsbR基因与花生、大豆编码氨基酸具有较高的同源性;根据MpsbR基因的氨基酸序列,与其它20个物种的氨基酸同源序列构建的系统进化树表明,桑树与花生、海桑、梨、大豆的亲缘关系较近。半定量RT-PCR研究表明,桑树光合系统MpsbR基因在桑树不同部位的表达水平有一定差异,在幼叶(刚展开第一张叶)和顶芽表达量最高,其作用机理有待于进一步研究之中。更多还原

【Abstract】 Mulberry (Morus alba) is an economically important plant used for sericulture. our country has the most abundant mulberry germplasm resources. The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses. Efforts to investigate the functional genes and their molecular adaptation mechanisms of stresses and to strengthen stress tolerance in this plant are of fundamental importance to the preservation of these genetic resources and to improve the yield of mulberry. However, the research of the functional genes from mulberry is in a backward state in comparison with other plants, and the exact function of some functional genes and their encoded proteins in the stress response in mulberry is still not fully understood yet. Therefore, the research of functional genes in mulberry is one urgent and the meaningful work.As a first step towards characterization of functional genes in mulberry, in this paper, we constructed a cDNA library from young mulberry leaves. Based on the research above, we cloned three important genes and carried out the analysis of their sequences. Furthermore, we conducted the functional verification of the three genes by the means of stress-induced method based on the prediction of their functions.The main results were summarized briefly as follows:1、EST encoding Chitinase was isolated from the cDNA library. And it’s full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for Chitinase in mulberry was designated M-chitinase (GenBank accession number: HQ117891). Sequence analysis showed that the M-chitinase is 1392 bp long and contains a 60 bp 5’-UTR (untranslated region) and a 255 bp 3’-UTR. It’s opening frame(ORF) is of 1077 bp,encoding 358 amino acids with apredicted molecular weight of 38.52KD and an isoelectric point of 4.466. Homology analysis revealed that M-chitinase gene are highly conservative in mulberry and other species including N. khasiana, Zea mays and Zea Diploperennis. Phylogenetic analysis based on M-chitinase gene with other 19 species showed that mulberry shows closer relationship with Nicotiana gossei, nicotiana tabacum, Capsicum annuum and rock cress. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-chitinase mRNA in the young leaf. And the transcriptional level of M-chitinase mRNA changed significantly under the conditions of cold, SA and ABA stresses respectively compared to the normal growth environment. 2、EST encoding PGIP was isolated from the cDNA library. And it’s full open reading frame were obtained by RACE and RT-PCR for the first time. A full length cDNA sequence coding for PGIP in mulberry was designated M-PGIP (GenBank accession number: HM044383). Sequence analysis showed that the M-PGIP is 1274bp long and contains a 93bp 5’-UTR (untranslated region) and a 179bp 3’-UTR.It’s opening frame(ORF) is of 1002bp,encoding 333 amino acids with apredicted molecular weight of 37.29KD and an isoelectric point of 7.25. Homology analysis revealed that M-PGIP gene are highly conservative in mulberry and other species including Prunus americana, Prunus persica and Prunus mume. Phylogenetic analysis based on M-PGIP gene with other 19 species showed that mulberry shows closer relationship with Cucumis melo, Vitis labrusca x Vitis riparia, Vitis vinifera and Actinidia deliciosa. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of M-PGIP mRNA in the young leaf. And the transcriptional level of M-PGIP mRNA changed significantly under the conditions of SA,ABA and salt sresses respectively compared to the normal growth environment.And we had expressed the recombinant protein to lay a good foundation for disease resistance Engineering of transgenic plants.3、The full length cDNA sequence coding for photosynthesis system II PsbR in mulberry Using the expression sequence label (expressed sequence tags, ESTs)was adopted the RT-PCR method to clone ,and named MpsbR (the Genbank accession number:GU937873 ). The sequential analysis indicated that this gene is 623bp long, and contains a 56bp 5 ’ - UTR and a 306bp 3 ’– UTR.It’s opening reading frame (ORF) is of 261bp, encoding 86 amino acids with apredicted molecular weight of 8.95KD and an isoelectric point of 11.09. Homology analysis revealed that , MpsbR gene are highly conservative in mulberry and other species including peanut, soybean and Prunus mume.; The phylogenetic analysis based on MpsbR gene with other 20 species showed that mulberry shows closer relationship with peanut, sea mulberry, pear and soybean. The results of semi quantitative RT-PCR analysis showed that the transcriptional level of MpsbR mRNA in the young leaf and the top bud. Its action mechanism is waiting for further studies.更多还原