本文選題：HECT　切入點(diǎn)：GhUPL7　出處：《西南大學(xué)》2016年碩士論文

【摘要】：UPL7是一類HECT(Homologous to the E6-associated protein carboxyl terminus)泛素連接酶,是泛素化途徑中一個(gè)特異性酶,與植物生長發(fā)育密切相關(guān)。為了解析UPL7在棉花纖維發(fā)育中的功能和作用機(jī)制,課題組前期從棉花中克隆了GhUPL7。本文首先分析和鑒定出陸地棉中的HECT基因,并描繪它們的結(jié)構(gòu)特征。試驗(yàn)獲得GhUPL7的表達(dá)特征以及其上游序列在植物中的表達(dá)模式,檢測和觀察反義抑制轉(zhuǎn)基因植株的胚珠、纖維和葉片表皮毛的變化,從而了解GhUPL7對(duì)棉花部分器官生長發(fā)育的影響,具體結(jié)果如下:1.陸地棉中的HECT基因進(jìn)化分析根據(jù)HECT序列從陸地棉數(shù)據(jù)庫檢索出HECT蛋白序列在棉花中的25個(gè)同源基因,即HECT基因。25個(gè)陸地棉HECT基因與擬南芥UPLs進(jìn)行進(jìn)化樹分析,可將GhUPLs歸類為4個(gè)分支,即亞家族I,II,III,IV。其中Gh_A01G1811和Gh_D01G2049兩個(gè)基因與克隆的GhUPL7基因相似度分別達(dá)到99%和98%,且基因Gh_A01G1811和Gh_D01G2049位于亞家族IV,與擬南芥AtUPL7親緣關(guān)系最近。為更好地理解不同HECT基因的結(jié)構(gòu)差異,根據(jù)它們的系統(tǒng)發(fā)生關(guān)系,分別將陸地棉基因組HECT蛋白結(jié)構(gòu)域和HECT序列的外顯子-內(nèi)含子結(jié)構(gòu)進(jìn)行比較。結(jié)果顯示:除C端的HECT結(jié)構(gòu)域外,陸地棉HECT基因在N端包含了其他的結(jié)構(gòu)域,推測這些結(jié)構(gòu)域決定HECT蛋白與不同底物相互作用。HECT亞家族IV的N端沒有結(jié)構(gòu)域或存在未知結(jié)構(gòu)域。關(guān)系密切的HECT基因在基因結(jié)構(gòu)上也更相似,尤其是在外顯子和內(nèi)含子的數(shù)量上,主要的不同是它們各自的外顯子-內(nèi)含子長度。以上分析表明GhUPL7確實(shí)是HECT類泛素連接酶,并且在進(jìn)化上具有保守性。2.GhUPL7的表達(dá)特征分析設(shè)計(jì)ghupl7特異性real-timepcr引物,檢測ghupl7基因在不同器官的表達(dá)水平發(fā)現(xiàn),其在柱頭中表達(dá)量相對(duì)較高,而在其它器官中表達(dá)量很低。檢測ghupl7基因在纖維不同時(shí)期的表達(dá)水平發(fā)現(xiàn),纖維快速伸長期的表達(dá)量相對(duì)較高。3.ghupl7上游序列的克隆及分析克隆ghupl7上游2189bp序列,并運(yùn)用植物啟動(dòng)子順式作用元件數(shù)據(jù)庫plantcare對(duì)ghupl7基因上游調(diào)控序列順式作用元件數(shù)量和種類進(jìn)行統(tǒng)計(jì)和匯總,結(jié)果顯示存在激素反應(yīng)相關(guān)元件,光反應(yīng)相關(guān)的元件,組織特異性表達(dá)的元件、脅迫反應(yīng)相關(guān)元件,結(jié)合位點(diǎn)和未知功能元件�？梢钥闯鲞@些順式作用元件與植物生長發(fā)育密切相關(guān)。繼而構(gòu)建啟動(dòng)子表達(dá)載體,煙草遺傳轉(zhuǎn)化,在煙草器官gus染色結(jié)果顯示:ghupl7啟動(dòng)子指導(dǎo)的gus基因主要在煙草柱頭中表達(dá),其次是花托、胚珠表面。進(jìn)一步檢測ghupl7啟動(dòng)子轉(zhuǎn)基因煙草不同組織的gus活性:在柱頭中酶活力最強(qiáng),卻僅有76.39nmol/min/mg,而不同時(shí)期的花托和子房相對(duì)較低,8dpa的花托和子房酶活力分別為11.13nmol/min/mg和10.28nmol/min/mg。以上結(jié)果表明ghupl7啟動(dòng)子指導(dǎo)的gus可能在花器官中表達(dá)。4.反義抑制ghupl7基因植株的種子增大,子指增加ghupl7是泛素化反應(yīng)中一個(gè)特異性酶,而泛素化反應(yīng)與植物生長發(fā)育息息相關(guān)。為探究該基因是否通過泛素化反應(yīng)參與三大營養(yǎng)物質(zhì)的代謝途徑,構(gòu)建植物表達(dá)載體camv35s::antisenseghupl7轉(zhuǎn)入陸地棉(冀棉14)中,并通過設(shè)計(jì)rt引物進(jìn)行real-timepcr篩選獲得反義抑制ghupl7的轉(zhuǎn)基因植株。收獲繁殖繼續(xù)篩選出t1代6個(gè)轉(zhuǎn)化子共42株植株,對(duì)10dpa胚珠的總氮和可溶性總糖含量進(jìn)行測定。結(jié)果顯示:總氮含量最高增加26.60%;可溶性總糖含量最高增加32.96%。與之對(duì)應(yīng)胚珠最后形成的種子增大,這與upl7擬南芥突變體表型相一致;產(chǎn)量性狀統(tǒng)計(jì)發(fā)現(xiàn):子指增加最高達(dá)14.86%。推測ghupl7可能參與泛素蛋白酶體途徑,影響碳氮代謝。5.反義抑制ghupl7基因植株短絨增加,衣分無明顯變化纖維的營養(yǎng)由胚珠供給,欲知反義抑制ghupl7對(duì)纖維發(fā)育的影響,對(duì)反義抑制ghupl7植株t1代進(jìn)行纖維起始觀察、糖含量測定、成熟纖維表型觀察、產(chǎn)量性狀統(tǒng)計(jì)。電鏡觀察反義抑制ghupl7基因?qū)γ藁ɡw維起始的影響,結(jié)果顯示纖維細(xì)胞起始出現(xiàn)異常纖維,但纖維的突起數(shù)量卻無明顯變化。糖含量測定顯示反義抑制ghupl7植株纖維可溶性總糖最高降低了30.66%;成熟纖維觀察結(jié)果顯示反義抑制ghupl7棉花長纖維不變或略變少,短絨增多。產(chǎn)量性狀統(tǒng)計(jì)結(jié)果表明衣分變化不顯著,最高降低了7.16%。6.反義抑制GhUPL7基因增加棉花葉片表皮毛的分支有研究報(bào)道UPLs家族的UPL3在擬南芥中與表皮毛的發(fā)育有關(guān),故而對(duì)同為HECT家族的GhUPL7反義抑制植株葉片表皮毛進(jìn)行觀察,并對(duì)其分支數(shù)進(jìn)行統(tǒng)計(jì),結(jié)果顯示1-3個(gè)分支的百分比明顯減少,大于4個(gè)分支的百分比明顯增多。該結(jié)果與upl7擬南芥突變體表皮毛分支數(shù)表型一致。反義抑制GhUPL7基因增加棉花葉片表皮毛的分支,推測表皮毛與纖維短絨發(fā)育具有相似機(jī)制。
[Abstract]:UPL7 is a kind of HECT (Homologous to the E6-associated protein carboxyl terminus) ubiquitin ligase is an enzyme specific ubiquitin pathway in plant growth and development are closely related to the function and mechanism of UPL7 on the development of cotton fiber, the research group before the period from cotton GhUPL7. was cloned firstly, analysis and identification the HECT gene in upland cotton, and describe their structural characteristics. Test the expression character of GhUPL7 and its upstream sequence in plant expression pattern detection and observation in antisense transgenic plants of ovule, fiber and leaf trichomes, so as to understand the effect of GhUPL7 on the growth and development of cotton organs, specific the results are as follows: 1. the evolution of the HECT gene in Upland Cotton analysis according to the sequence of HECT from the database to retrieve the upland cotton HECT protein sequence of 25 homologues in the cotton, namely HEC Phylogenetic analysis of T gene.25 in Upland Cotton HECT gene of Arabidopsis UPLs, GhUPLs can be classified into 4 branches, namely the subfamily I, II, III, IV., Gh_A01G1811 and Gh_D01G2049 GhUPL7 gene similarity of two genes and cloning were respectively 99% and 98%, and the Gh_A01G1811 and Gh_D01G2049 genes in the IV subfamily. The relationship between AtUPL7 and Arabidopsis genetic recently. In order to better understand the different structure of the HECT gene, according to their phylogeny, the upland cotton genome HECT protein domain HECT sequence and exon intron structure were compared. The results showed that except for the HECT domain of the C, HECT gene in upland cotton contains the other domain at the N end, that these domains determine the HECT protein with different substrate interaction.HECT subfamily IV N terminal is not domain or unknown domain. Close HECT gene at base Because the structure is more similar, especially the number of exons and introns in and abroad, the main difference is their exon intron length. The above analysis shows that GhUPL7 is indeed HECT ubiquitin ligase, expression and characteristic of evolutionarily conserved.2.GhUPL7 analysis and design of ghupl7 specific real-timepcr primers. Detection of ghupl7 gene expression levels found in different organs, the expression was relatively high in the stigma, and in other organs in the expression is very low. The detection of ghupl7 gene expression level found in different periods of fiber, fiber rapidly extending long-term relatively high expression of.3.ghupl7 upstream sequence cloning and analysis of cloned ghupl7 upstream 2189bp sequence. And the use of plant promoter CIS element database of plantcare ghupl7 gene upstream regulation sequence along the number and type of elements of statistics and a summary of the results Related components showed the presence of hormone response, light responsive element, the element of the tissue-specific expression of stress response element binding sites and unknown functional elements. It can be seen that these cis elements to plant growth and development are closely related. And then construct a promoter expression vector, tobacco genetic transformation in tobacco organs Gus staining display: GUS gene ghupl7 promoter directed mainly expressed in tobacco stigma, followed by the receptacle, ovule surface. Further detection of ghupl7 promoter in different tissues of transgenic tobacco sub GUS activity: the stigma of enzyme activity in the strongest, but only 76.39nmol/min/mg, and in different periods of the receptacle and ovary is relatively low, the 8dpa receptacle and ovary enzyme activity was above 11.13nmol/min/mg and 10.28nmol/min/mg. results show that the ghupl7 promoter directed.4. antisense Gus may inhibit ghupl7 gene expression in floral organs A result of the increase in plant seeds, sub refers to the increase in ghupl7 is a specific enzyme ubiquitination reaction, and ubiquitination reaction to plant growth and development is closely related to metabolic pathways. Explore whether the gene by ubiquitination reaction in three major nutrients, construction of plant expression vector camv35s:: antisenseghupl7 into Upland Cotton (Jimian 14), and the design of RT primers for real-timepcr screening to obtain transgenic plants with antisense inhibition of ghupl7. Screening of T1 generation breeding to harvest 6 transformants of 42 plants, the total sugar content of total nitrogen and soluble in 10dpa ovules were determined. The results showed that the highest content of total nitrogen increased by 26.60%; the highest content of total soluble sugar with the increase of corresponding 32.96%. ovule finally formed seeds increased, and the upl7 Arabidopsis mutant found consistent; yield statistics: refers to the increase of up to 14.86%. Ghupl7 can speculate To participate in the ubiquitin proteasome pathway, carbon and nitrogen metabolism of.5. antisense inhibition of ghupl7 transgenic plants increased lint percentage had no significant change in short fiber, fiber nutrition from ovule supply, to know the antisense inhibition effect of ghupl7 on the development of fiber, the antisense inhibition of ghupl7 plants of T1 generation of fiber was observed, determination of sugar content in mature fiber. The phenotype observation, yield statistics. Electron microscope was used to observe the effects of antisense suppression of ghupl7 gene on cotton fiber initiation, the result shows that the fiber cell initiation abnormal fiber, but the number of fiber projections had no obvious changes. Sugar content measurement showed that antisense inhibition of ghupl7 plant fiber soluble sugar reduced up to 30.66%; the mature fiber observation results show that anti suppression of ghupl7 cotton fiber length unchanged or slightly less, linter increased. Statistical results yield traits showed that lint percentage did not change significantly, the highest was reduced by 7.16%.6. antisense inhibition of GhUP The L7 gene of cotton leaf epidermal hair increased branch studies have reported UPLs family UPL3 in Arabidopsis and epidermal hair development. Therefore, to observe the same as the family of HECT GhUPL7 antisense inhibition of leaf epidermal hair, and ststisticed the branch number, results show that the percentage of 1-3 branches decreased significantly greater than the percentage the 4 branches increased significantly. The results of upl7 and Arabidopsis mutant trichome branches phenotype is consistent. Antisense inhibition of GhUPL7 gene in cotton leaf epidermis increased branching hair, presumably trichomes and fiber has similar fuzz development mechanism.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2;S562

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