本文選題：柿 + SSAP　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：柿(Diospyros kaki Thunb.;2n=6x=90)原產(chǎn)中國(guó),是柿屬植物中最具經(jīng)濟(jì)價(jià)值的栽培種,我國(guó)柿栽培面積和產(chǎn)量均居世界第一。但傳統(tǒng)產(chǎn)區(qū)多為完全澀柿(PCA),需人工脫澀方能食用;而完全甜柿(PCNA)在樹上可自然脫澀,且其果實(shí)貨架期較長(zhǎng),更具商品價(jià)值,是未來(lái)柿育種的重要目標(biāo)。已經(jīng)明確,中國(guó)原產(chǎn)完全甜柿(簡(jiǎn)稱“中國(guó)甜柿”或C-PCNA)自然脫澀性狀受顯性單基因位點(diǎn)控制,因而在完全甜柿的遺傳改良中具有重大應(yīng)用潛力。前期研究表明,中國(guó)甜柿自然脫澀可能涉及可溶性單寧的自然凝固過(guò)程,但其機(jī)理尚不完全清楚。本文以中國(guó)甜柿為試材,首先利用SSAP技術(shù)構(gòu)建溫水脫澀差異表達(dá)cDNA文庫(kù),獲得中國(guó)甜柿溫水脫澀過(guò)程中與“凝固效應(yīng)”相關(guān)的候選差異基因片段,同時(shí)結(jié)合非完全甜柿(non-PCNA)對(duì)溫水脫澀機(jī)理進(jìn)行初步探討;其次,基于cDNA-SSAP文庫(kù)篩選參與中國(guó)甜柿的自然脫澀的差異轉(zhuǎn)錄片段,通過(guò)RACE及染色體步移獲取其cDNA全長(zhǎng),并利用柿葉片瞬時(shí)超表達(dá)技術(shù)對(duì)候選基因功能驗(yàn)證;最后,應(yīng)用酵母單雜交系統(tǒng)篩選與脫澀基因互作的候選轉(zhuǎn)錄因子,進(jìn)一步探討中國(guó)甜柿自然脫澀的調(diào)控機(jī)理。本研究為中國(guó)甜柿可溶性單寧自然凝固的分子機(jī)理以及甜柿遺傳改良研究提供科學(xué)依據(jù)。主要研究結(jié)果如下:1.應(yīng)用cDNA-SSAP技術(shù)構(gòu)建中國(guó)甜柿溫水脫澀前后差異轉(zhuǎn)錄的cDNA文庫(kù),經(jīng)qRT-PCR分析及轉(zhuǎn)錄組數(shù)據(jù)庫(kù)比對(duì)確認(rèn)了文庫(kù)數(shù)據(jù)的可靠性及準(zhǔn)確性。cDNA文庫(kù)包含差異轉(zhuǎn)錄條帶283條,Blast2GO分析顯示其主要參與單寧合成、跨膜轉(zhuǎn)運(yùn)、糖酵解和果膠代謝等不同的生物學(xué)過(guò)程。2.醛類介導(dǎo)的“凝固效應(yīng)”及果膠的累積是柿果溫水脫澀的主要原因�；诓町惐磉_(dá)cDNA文庫(kù)新發(fā)現(xiàn)3條參與醛類代謝候選基因片段,1條參與果膠代謝的差異轉(zhuǎn)錄片段,qRT-PCR分析顯示其在中國(guó)甜柿和非完全甜柿溫水脫澀過(guò)程中均上調(diào)表達(dá)。結(jié)合單寧及水溶性果膠含量測(cè)定,結(jié)果表明醛類及果膠的累積是柿果溫水脫澀主要原因。3.基于cDNA-SSAP文庫(kù),篩選得到參與中國(guó)甜柿自然脫澀的候選基因片段TDF 284-2(PK基因)(轉(zhuǎn)錄條帶;transcript-derived fragment;TDF),利用RACE及Walking技術(shù)首次在柿屬植物上分離獲得6條DkPK的cDNA全長(zhǎng)。4.DkPK1潛在參與中國(guó)甜柿的自然脫澀。分析6條DkPK基因在不同脫澀類型柿果實(shí)發(fā)育過(guò)程中的表達(dá)模式及其與可溶性單寧的關(guān)系,結(jié)果表明DkPK1表達(dá)變化與中國(guó)甜柿可溶性單寧含量呈高度負(fù)相關(guān);聚類分析及亞細(xì)胞定位顯示其為細(xì)胞質(zhì)PK,潛在參與調(diào)控及醛類的代謝。瞬時(shí)超表達(dá)DkPK1可顯著降低柿葉片中可溶性單寧含量,同時(shí)顯著上調(diào)其下游醛類代謝關(guān)鍵基因DkPDC1,3,4,5和DkADH1,3表達(dá)量。由此推測(cè)DkPK1的上調(diào)表達(dá)可促進(jìn)可溶性單寧的“凝固”進(jìn)而導(dǎo)致中國(guó)甜柿的自然脫澀,并提出中國(guó)甜柿自然脫澀分子機(jī)制模型圖。5.分離得到4個(gè)DkPK1的上游調(diào)控因子�；贒kPK1全長(zhǎng)序列信息,通過(guò)染色體步移分離得到2,000 bp左右的啟動(dòng)子序列。應(yīng)用酵母單雜交技術(shù)從酵母文庫(kù)中篩選分離140個(gè)目的單克隆,最終獲得4個(gè)與DkPK1互作的上游調(diào)控因子,分別為MADS-Box、WRKY、MYB和YABBY類轉(zhuǎn)錄因子。此外,利用同樣方法分離得到3個(gè)與已知基因DkCAD1互作的候選轉(zhuǎn)錄因子。綜上所述,基于SSAP技術(shù),我們構(gòu)建了中國(guó)甜柿柿果溫水脫澀前后差異表達(dá)cDNA文庫(kù),結(jié)合對(duì)非完全甜柿的分析,初步闡明乙醛介導(dǎo)的“凝固效應(yīng)”及果膠-單寧復(fù)合物的形成是導(dǎo)致柿果溫水脫澀主要原因;分離鑒定6個(gè)DkPK基因,初步確定DkPK1參與了中國(guó)甜柿的自然脫澀;最后,通過(guò)酵母單雜交技術(shù)在酵母文庫(kù)中分離4個(gè)調(diào)控DkPK1的候選轉(zhuǎn)錄因子,其中3個(gè)轉(zhuǎn)錄因子在柿中系首次報(bào)道。中國(guó)甜柿自然脫澀相關(guān)基因及轉(zhuǎn)錄因子的獲取,為甜柿自然脫澀調(diào)控機(jī)理研究和未來(lái)完全甜柿新種質(zhì)的創(chuàng)造提供科學(xué)依據(jù)并奠定分子育種技術(shù)基礎(chǔ)。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S665.2

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