中国甜柿cDNA-SSAP文库构建及自然脱涩相关基因功能验证

发布时间：2018-04-26 03:05

本文选题：柿 + SSAP　；参考：《华中农业大学》2016年博士论文

【摘要】：柿(Diospyros kaki Thunb.;2n=6x=90)原产中国,是柿属植物中最具经济价值的栽培种,我国柿栽培面积和产量均居世界第一。但传统产区多为完全涩柿(PCA),需人工脱涩方能食用;而完全甜柿(PCNA)在树上可自然脱涩,且其果实货架期较长,更具商品价值,是未来柿育种的重要目标。已经明确,中国原产完全甜柿(简称“中国甜柿”或C-PCNA)自然脱涩性状受显性单基因位点控制,因而在完全甜柿的遗传改良中具有重大应用潜力。前期研究表明,中国甜柿自然脱涩可能涉及可溶性单宁的自然凝固过程,但其机理尚不完全清楚。本文以中国甜柿为试材,首先利用SSAP技术构建温水脱涩差异表达cDNA文库,获得中国甜柿温水脱涩过程中与“凝固效应”相关的候选差异基因片段,同时结合非完全甜柿(non-PCNA)对温水脱涩机理进行初步探讨;其次,基于cDNA-SSAP文库筛选参与中国甜柿的自然脱涩的差异转录片段,通过RACE及染色体步移获取其cDNA全长,并利用柿叶片瞬时超表达技术对候选基因功能验证;最后,应用酵母单杂交系统筛选与脱涩基因互作的候选转录因子,进一步探讨中国甜柿自然脱涩的调控机理。本研究为中国甜柿可溶性单宁自然凝固的分子机理以及甜柿遗传改良研究提供科学依据。主要研究结果如下:1.应用cDNA-SSAP技术构建中国甜柿温水脱涩前后差异转录的cDNA文库,经qRT-PCR分析及转录组数据库比对确认了文库数据的可靠性及准确性。cDNA文库包含差异转录条带283条,Blast2GO分析显示其主要参与单宁合成、跨膜转运、糖酵解和果胶代谢等不同的生物学过程。2.醛类介导的“凝固效应”及果胶的累积是柿果温水脱涩的主要原因。基于差异表达cDNA文库新发现3条参与醛类代谢候选基因片段,1条参与果胶代谢的差异转录片段,qRT-PCR分析显示其在中国甜柿和非完全甜柿温水脱涩过程中均上调表达。结合单宁及水溶性果胶含量测定,结果表明醛类及果胶的累积是柿果温水脱涩主要原因。3.基于cDNA-SSAP文库,筛选得到参与中国甜柿自然脱涩的候选基因片段TDF 284-2(PK基因)(转录条带;transcript-derived fragment;TDF),利用RACE及Walking技术首次在柿属植物上分离获得6条DkPK的cDNA全长。4.DkPK1潜在参与中国甜柿的自然脱涩。分析6条DkPK基因在不同脱涩类型柿果实发育过程中的表达模式及其与可溶性单宁的关系,结果表明DkPK1表达变化与中国甜柿可溶性单宁含量呈高度负相关;聚类分析及亚细胞定位显示其为细胞质PK,潜在参与调控及醛类的代谢。瞬时超表达DkPK1可显著降低柿叶片中可溶性单宁含量,同时显著上调其下游醛类代谢关键基因DkPDC1,3,4,5和DkADH1,3表达量。由此推测DkPK1的上调表达可促进可溶性单宁的“凝固”进而导致中国甜柿的自然脱涩,并提出中国甜柿自然脱涩分子机制模型图。5.分离得到4个DkPK1的上游调控因子。基于DkPK1全长序列信息,通过染色体步移分离得到2,000 bp左右的启动子序列。应用酵母单杂交技术从酵母文库中筛选分离140个目的单克隆,最终获得4个与DkPK1互作的上游调控因子,分别为MADS-Box、WRKY、MYB和YABBY类转录因子。此外,利用同样方法分离得到3个与已知基因DkCAD1互作的候选转录因子。综上所述,基于SSAP技术,我们构建了中国甜柿柿果温水脱涩前后差异表达cDNA文库,结合对非完全甜柿的分析,初步阐明乙醛介导的“凝固效应”及果胶-单宁复合物的形成是导致柿果温水脱涩主要原因;分离鉴定6个DkPK基因,初步确定DkPK1参与了中国甜柿的自然脱涩;最后,通过酵母单杂交技术在酵母文库中分离4个调控DkPK1的候选转录因子,其中3个转录因子在柿中系首次报道。中国甜柿自然脱涩相关基因及转录因子的获取,为甜柿自然脱涩调控机理研究和未来完全甜柿新种质的创造提供科学依据并奠定分子育种技术基础。
[Abstract]:Persimmon (Diospyros kaki Thunb.; 2n=6x=90), native to China, is the most economical cultivated species of persimmon plants. The cultivated area and yield of persimmon are the first in the world. But the traditional producing area is mostly astringent persimmon (PCA), which needs to be consumed artificially, and the complete persimmon (PCNA) can be naturally unastringent in the tree, and the shelf life of the fruit is longer and has a more commodity. Value is an important goal in the future of persimmon breeding. It is clear that natural persimmon ("Chinese persimmon" or C-PCNA) natural disastringent traits in China are controlled by dominant single gene loci, and therefore have great potential in the genetic improvement of complete persimmon. Natural solidification process, but its mechanism is not completely clear. In this paper, Chinese persimmon was used as a test material. First, SSAP technology was used to construct the differential expression cDNA Library of warm water, and the candidate differential gene fragments related to "coagulation effect" in the process of warm water removal in Chinese persimmon were obtained. At the same time, the mechanism of non complete persimmons (non-PCNA) was joined to the mechanism of warm water acerbity. Secondly, based on the cDNA-SSAP library, we screened the naturally acerbic transcriptional fragments of Chinese sweet persimmons, obtained the full length of cDNA through RACE and chromosome steps, and verified the function of the candidate genes by the instantaneous overexpression of persimmon leaf slices. Finally, the yeast single hybrid system was used to screen the candidate transcriptional causes of the interactivity of the hyper astringent gene. This study provides a scientific basis for the molecular mechanism of natural solidification of soluble tannins in Chinese persimmon and the genetic improvement of sweet persimmons. The main results are as follows: 1. the cDNA Library of different transcriptional transcription before and after the warm water of Chinese persimmons was constructed by cDNA-SSAP technology, and the qRT-PCR score was divided into two parts. Analysis and transcriptional database comparison confirm the reliability and accuracy of the library data. The.CDNA library contains 283 different transcriptional strips. Blast2GO analysis shows that it is mainly involved in tannin synthesis, transmembrane transport, glycolysis and pectin metabolism, and the "coagulation effect" mediated by.2. aldehydes and the accumulation of pectin are the warm water of persimmon fruit. Based on the difference expression cDNA library, 3 new genes were found to participate in the aldehyde metabolism candidate gene fragments, and 1 were involved in the differential transcriptional fragments of pectin metabolism. QRT-PCR analysis showed that it was up to up expression in the process of warm water disastringency of Chinese persimmon and non complete persimmon. The accumulation of pectin and pectin is the main cause of persimmon fruit temperature water acerbity main reason.3. based on cDNA-SSAP library, the candidate gene fragment TDF 284-2 (PK gene) (PK gene) (transcriptional strip; transcript-derived fragment; TDF), which participates in the natural acerbity of Chinese persimmon, is selected for the first time to separate 6 DkPK cDNA full-length.4.DkPK1 from the persimmon plants by RACE and Walking technology. The expression patterns of 6 DkPK genes in the development of different persimmons and their relationship with soluble tannins were analyzed. The results showed that the change of DkPK1 expression was negatively correlated with the content of soluble tannin in Chinese persimmon; cluster analysis and subcellular localization showed that it was a cytoplasmic PK. The content of soluble tannin in persimmon leaf slices was significantly reduced by transient overexpression of DkPK1, and the expression of key genes, DkPDC1,3,4,5 and DkADH1,3, was significantly up-regulated in the leaves of persimmon leaves. Therefore, the up-regulated expression of DkPK1 could promote the "coagulation" of soluble tannin and lead to the natural disastringent of Chinese persimmon. The molecular mechanism model of natural persimmons in Chinese persimmons.5. was separated to get the upstream regulator of 4 DkPK1. Based on the information of DkPK1 full length sequence, 2000 BP promoter sequences were separated by chromosome step separation. The yeast single hybridization technique was used to screen and separate 140 targets from the yeast library. Finally, 4 DkPK1 interactions were obtained. The upstream regulators are MADS-Box, WRKY, MYB and YABBY transcription factors, respectively. In addition, 3 candidate transcriptional factors that interact with known gene DkCAD1 are obtained by the same method. In summary, based on SSAP technology, we constructed the differential expression cDNA Library of Chinese persimmon persimmon fruit and persimmon fruit before and after the warm water, combined with the analysis of the non complete persimmon. It is preliminarily elucidated that acetaldehyde mediated "coagulation effect" and the formation of pectin - tannin complex are the main causes of persimmon fruit warm water acerbity. 6 DkPK genes were isolated and identified, and DkPK1 was preliminarily determined to be involved in the natural acerbity of Chinese persimmon. Finally, 4 candidate transcription factors regulating DkPK1 were separated by yeast single hybridization in the yeast library. The first 3 transcriptional factors are reported in the persimmon line for the first time. The acquisition of natural depastringrelated genes and transcription factors in Chinese persimmons provides scientific basis for the study of natural dislocation regulation mechanism of persimmon and the creation of new germplasm of complete persimmon in the future, and lays the foundation for molecular breeding technology.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S665.2

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