【摘要】：不同植物對(duì)光周期響應(yīng)不同,目前關(guān)于植物如何在分子水平響應(yīng)光周期知之甚少。本論文以不同光周期處理的馬鈴薯葉片為材料,轉(zhuǎn)錄組測(cè)序獲得了不同光周期處理的轉(zhuǎn)錄組表達(dá)譜;分析了表達(dá)譜數(shù)據(jù),識(shí)別和篩選了光周期響應(yīng)基因,克隆了光周期響應(yīng)基因StH2A的全長(zhǎng)cDNA,基因工程途徑實(shí)現(xiàn)了反義StH2A cDNA在本氏煙體內(nèi)表達(dá),確定了 H2A參與頂端優(yōu)勢(shì)維持和調(diào)控開(kāi)花的功能,具體研究結(jié)果如下:1.經(jīng)轉(zhuǎn)錄組測(cè)序、GO功能注釋和差異表達(dá)基因分析,篩選了 208個(gè)注釋為“核酸結(jié)合蛋白”的差異基因。結(jié)合不同光周期這些基因的RPKM表達(dá)量變化,篩選了 20個(gè)顯著隨光周期變化表達(dá)的目的基因,qRT-PCR證實(shí)了其隨光周期的表達(dá)動(dòng)態(tài),由此確定了對(duì)注釋為H3.2、H2A和TP的基因進(jìn)行進(jìn)一步研究。2.克隆了H3.2、H2A、TP基因的全長(zhǎng)cDNA;為了實(shí)現(xiàn)各自編碼基因體內(nèi)過(guò)量或抑制表達(dá),亞克隆途徑,得到基因工程重組菌株 DH5α-pC-35S-H3.2+/-、DH5α-pC-35S-H2A+/-、DH5α-pC-35S-TP+/-及GV3101-pC-35S-H3.2+/-、GV3101-pC-35S-H2A+/-、GV3101-pC-35S-TP+/-。3.采用根癌農(nóng)桿菌介導(dǎo)途徑,轉(zhuǎn)化反義StH2A于本氏煙,篩選獲得本氏煙突變株系h2a-2,h2a-2表現(xiàn)出頂端優(yōu)勢(shì)缺失和開(kāi)花延遲的表型;H2A蛋白ELISA測(cè)定結(jié)果顯示,h2a-2株系葉片H2A蛋白含量比WT下降23.7%,表明反義StH2A本氏煙體內(nèi)表達(dá)抑制了本氏煙NbH2A的積累。4.本氏煙基因組數(shù)據(jù)庫(kù)檢索,識(shí)別了CONSTANS(CO),FLOWING LOCUS T(FT)和EARLY FLOWERING 4(LF4)3個(gè)成花基因,依據(jù)其核酸序列設(shè)計(jì)各自特異引物,葉片mRNA為模版,RT-PCR結(jié)果顯示,與對(duì)照株系比較,h2a-2株系3個(gè)成花基因mRNA積累呈現(xiàn)不同程度的下調(diào)表達(dá),表明NbH2A參與了 3個(gè)成花基因的轉(zhuǎn)錄,CO,FT與ELF4間通過(guò)H2A彼此聯(lián)系。5.檢索本氏煙基因組數(shù)據(jù)庫(kù),識(shí)別了 22個(gè)NbH2A編碼基因,序列對(duì)比和進(jìn)化樹(shù)分析發(fā)現(xiàn),其中4個(gè)NbH2A與本研究克隆的StH2A預(yù)測(cè)的氨基酸序列高度相似,且具有N端多聚丙氨酸基序,反義StH2A至少抑制了 N端多聚丙氨酸NbH2A的表達(dá)。6.不同NbH2A基因RT-PCR結(jié)果顯示,h2a-2株系葉片多聚丙氨酸NbH2A mRNA積累明顯少于對(duì)照,其它非多聚丙氨酸NbH2A基因mRNA積累明顯多于對(duì)照,表明其它NbH2A基因在轉(zhuǎn)錄水平互補(bǔ)了多聚丙氨酸NbH2A的表達(dá)下調(diào)。結(jié)論認(rèn)為:反義StH2A在本氏煙體內(nèi)表達(dá),抑制了其多聚丙氨酸NbH2A基因mRNA積累,盡管其它非多聚丙氨酸NbH2A mRNA積累增加,并未能彌補(bǔ)煙草H2A蛋白積累的減少;煙草H2A蛋白積累減少,導(dǎo)致頂端優(yōu)勢(shì)缺失和開(kāi)花推遲的表型,說(shuō)明編碼多聚丙氨酸的NbH2A基因正向調(diào)控本氏煙的開(kāi)花和生長(zhǎng)。
[Abstract]:The response of different plants to photoperiod is different, and little is known about how plants respond to photoperiod at molecular level. In this paper, the transcriptome expression profiles of potato leaves treated with different photoperiod were obtained by transcriptome sequencing, and the data of expression profiles were analyzed, and the photocycle-responsive genes were identified and screened. The full-length cDNA of photoperiodic response gene (StH2A) was cloned. The antisense StH2A cDNA was expressed in the tobacco by genetic engineering pathway, and the function of H2A in maintaining and controlling flowering was confirmed. The results are as follows: 1. By sequencing go functional annotation and differential expression gene analysis, 208 differentially expressed genes annotated as "nucleic acid binding protein" were screened. In combination with the changes of RPKM expression of these genes in different photoperiod, 20 target genes were screened and their expression dynamics with photoperiod were confirmed by qRT-PCR. Therefore, further studies on the genes annotated as H3.2, H2A and TP have been carried out. The full-length cDNAof H3.2H2Atp gene was cloned, and the recombinant strain DH5 偽 -pC-35S-H3.2 / -DH5 偽 -pC-35S-H2A 偽 -pC-35S-TP /-and GV3101-pC-35S-H3.2 / -GV3101-pC-35S-H2A / -GV3101-pC-35S-TP / -3.3in order to achieve excessive or inhibitory expression in vivo were obtained from the recombinant strain DH5 偽 -pC-35S-H3.2% -pC-35S-H3.2 偽 -pC-35S-H2A 偽 -pC-35S-H2A-pC-35S-TP / -3. Agrobacterium tumefaciens mediated transformation of antisense StH2A into Bentner's tobacco was carried out by Agrobacterium tumefaciens. The phenotypic H2A protein ELISA analysis showed that the H2A protein content in leaves of H2a-2a-2 mutant was lower than that of WT, indicating that the in vivo expression of antisense StH2A inhibited the accumulation of NbH2A. 4. Three floral genes of CONSTANS (CO) flushing LOCUS T (FT) and EARLY FLOWERING 4 (LF4) were identified and their specific primers were designed according to their nucleic acid sequences. The results of RT-PCR showed that mRNA in leaves was a template. Compared with the control lines, the mRNA accumulation of three floral genes of H2a-2 strain was down-regulated in varying degrees, indicating that NbH2A was involved in the transcription of three floral genes, Cocoft and ELF4 were related to each other through H2A. 5. 22 NbH2A coding genes were identified by searching the database of tobacco genome. Sequence comparison and phylogenetic tree analysis showed that 4 of them were highly similar to the predicted amino acid sequences of the cloned StH2A and had N-terminal polyalanine motifs. Antisense StH2A inhibited at least the expression of N terminal polyalanine NbH2A. The results of RT-PCR of different NbH2A genes showed that the accumulation of polyalanine NbH2A mRNA in leaves of H2a-2 strains was significantly lower than that of the control, and the mRNA accumulation of other non-polyalanine NbH2A genes was significantly higher than that of the control. These results suggest that other NbH2A genes complement each other in down-regulation of polyalanine NbH2A expression at the transcriptional level. Conclusion: the expression of antisense StH2A in tobacco inhibits the mRNA accumulation of polyalanine NbH2A gene, although the accumulation of other non-polyalanine NbH2A mRNA increases, but it can not make up for the decrease of H2A protein accumulation and H2A protein accumulation in tobacco. The phenotypes of apical dominance loss and flowering delay indicate that the NbH2A gene encoding polyalanine positively regulates the flowering and growth of Bentworth tobacco.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2

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本文編號(hào)：2145059