【摘要】：NAC轉(zhuǎn)錄因子是植物特有的具有多種生物調(diào)控功能的轉(zhuǎn)錄因子。研究表明,NAC轉(zhuǎn)錄因子參與調(diào)控植物生長(zhǎng)發(fā)育、激素信號(hào)轉(zhuǎn)導(dǎo)途徑及脅迫應(yīng)答反應(yīng)。本文利用生物信息學(xué)的方法對(duì)番茄NAC4-9轉(zhuǎn)錄因子進(jìn)行結(jié)構(gòu)分析和生物功能預(yù)測(cè)。利用病毒誘導(dǎo)的基因沉默技術(shù)(VIGS)對(duì)NAC轉(zhuǎn)錄因子編碼基因及乙烯合成和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因進(jìn)行沉默,以此研究NAC轉(zhuǎn)錄因子在番茄成熟及乙烯信號(hào)轉(zhuǎn)導(dǎo)途徑中的調(diào)控作用。主要研究結(jié)果如下:(1)生物信息學(xué)分析:對(duì)SNAC4-9轉(zhuǎn)錄因子蛋白的高級(jí)結(jié)構(gòu)和生物調(diào)控功能預(yù)測(cè)結(jié)果顯示,SNAC4和SNAC6與擬南芥ANAC056最相近;SNAC5和SNAC8具有相似的高級(jí)結(jié)構(gòu)且與擬南芥ANAC072最相近;SNAC7和SNAC9與擬南芥ANAC029最相近且三者具有相似的三級(jí)結(jié)構(gòu)。生物調(diào)控功能預(yù)測(cè)結(jié)果顯示,SNAC4-9轉(zhuǎn)錄因子還可能參與調(diào)控生長(zhǎng)素、脫落酸、茉莉酸等植物激素的信號(hào)轉(zhuǎn)導(dǎo)途經(jīng)。分析乙烯合成和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因LeACS4、LeACO1、NR、LeETR4、LeCTR1、LeERF2的啟動(dòng)子序列進(jìn)行轉(zhuǎn)錄因子結(jié)合位點(diǎn)預(yù)測(cè),發(fā)現(xiàn)乙烯相關(guān)因子可能參與調(diào)控茉莉酸、脫落酸和水楊酸等激素響應(yīng)及高溫、干旱等逆境脅迫反應(yīng)。(2)VIGS技術(shù)對(duì)轉(zhuǎn)錄因子的生物功能研究:構(gòu)建pTRV2-LeACS4/LeACO1/NR/LeETR4/LeCTR1/LeERF2載體,應(yīng)用注射法侵染活體番茄植株,沉默番茄果實(shí)中的SNAC4/5/6/7/9和LeACS4/LeACO1/NR/LeETR4/LeCTR1/LeERF2/LeRIN基因。在農(nóng)桿菌侵染10天后,沉默SNACs基因的番茄植株株高低于對(duì)照組。沉默SNACs及乙烯相關(guān)基因的植株,番茄果實(shí)表現(xiàn)出成熟延遲表型,而只注射含空TRV2載體的農(nóng)桿菌菌液的對(duì)照組果實(shí)照常成熟。(3)基因沉默的番茄果實(shí)轉(zhuǎn)錄水平檢測(cè)結(jié)果顯示SNAC基因的沉默影響了番茄果實(shí)中乙烯合成和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因的表達(dá)情況。SNAC基因的沉默會(huì)導(dǎo)致乙烯合成基因LeACS2/LeACS4/LeACO1及乙烯受體基因NR/LeETR4表達(dá)水平降低。同時(shí),SNAC基因的表達(dá)水平也受到乙烯合成及信號(hào)轉(zhuǎn)導(dǎo)基因表達(dá)情況的影響,乙烯合成基因LeACS4/LeACO1和乙烯信號(hào)轉(zhuǎn)導(dǎo)相關(guān)因子LeCTR1/LeERF2的編碼基因的沉默抑制SNAC基因表達(dá),乙烯受體基因NR/LeETR4的沉默誘導(dǎo)SNAC基因表達(dá)水平升高。(4)沉默SNAC4基因后,在成熟過(guò)程中,番茄果實(shí)的乙烯釋放速率下降,并且延遲了乙烯釋放高峰的出現(xiàn)時(shí)間。
[Abstract]:NAC transcription factors are plant specific transcription factors with various biological regulatory functions. The results showed that NAC transcription factors were involved in plant growth and development, hormone signal transduction pathway and stress response. In this paper, bioinformatics was used to analyze the structure and biofunction of tomato NAC4-9 transcription factors. Viral induced gene silencing technique (VIGS) was used to silence NAC transcription factor encoding genes and ethylene synthesis and signal transduction related genes in order to study the regulatory role of NAC transcription factors in tomato maturation and ethylene signal transduction pathway. The main results are as follows: (1) Bioinformatics analysis: the prediction of the advanced structure and bioregulatory function of SNAC4-9 transcription factor protein shows that SNAC4 and SNAC6 have similar high-level structures to Arabidopsis ANAC056 and SNAC5 and SNAC8. SNAC7 and SNAC9 are the most similar in ANAC072 and ANAC029 in Arabidopsis thaliana, and the three have similar tertiary structures. The prediction of biological regulation function indicates that SNAC4-9 transcription factor may also be involved in the regulation of auxin, abscisic acid, jasmonic acid and other plant hormones signal transduction pathway. By analyzing the promoter sequence of LeACS4, LeACO1, LeETR4, LeCTR1, LeERF2, and predicting transcription factor binding sites, we found that ethylene related factors may be involved in regulating the response of hormones such as jasmonic acid, abscisic acid and salicylic acid and high temperature. (2) study on the biological function of transcription factors by VIGS: construct pTRV2-LeACS4/LeACO1/NR/LeETR4/LeCTR1/LeERF2 vector, infect tomato plants in vivo by injection method, silence the SNAC4/5/6/7/9 and LeACS4/LeACO1/NR/LeETR4/LeCTR1/LeERF2/LeRIN genes in tomato fruit. After infection with Agrobacterium tumefaciens for 10 days, the plant height of tomato with SNACs gene silencing was lower than that of control group. Tomato fruits with silencing SNACs and ethylene-related genes showed delayed maturation phenotype. The fruit of control group treated with Agrobacterium tumefaciens containing empty TRV2 vector was mature as usual. (3) the transcriptional level of tomato fruit with gene silencing showed that the silencing of SNAC gene affected ethylene synthesis and signal transduction in tomato fruit. The silencing of SNAC gene resulted in the decrease of LeACS2/LeACS4/LeACO1 and NR/LeETR4 expression of ethylene biosynthesis gene. At the same time, the expression level of SNAC gene was also affected by ethylene synthesis and signal transduction gene expression. The silencing of ethylene synthesis gene LeACS4/LeACO1 and the encoding gene of ethylene signal transduction related factor LeCTR1/LeERF2 inhibited the expression of SNAC gene. The silencing of ethylene receptor gene NR/LeETR4 induced the increase of SNAC gene expression. (4) after silencing the SNAC4 gene, the ethylene release rate of tomato fruit decreased during maturation, and the emergence time of ethylene release peak was delayed.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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本文編號(hào)：2184825