抑制棉花中GhUPL7基因表达影响胚珠和表皮毛发育

发布时间：2018-03-30 02:15

  本文选题：HECT　切入点：GhUPL7　出处：《西南大学》2016年硕士论文

【摘要】：UPL7是一类HECT(Homologous to the E6-associated protein carboxyl terminus)泛素连接酶,是泛素化途径中一个特异性酶,与植物生长发育密切相关。为了解析UPL7在棉花纤维发育中的功能和作用机制,课题组前期从棉花中克隆了GhUPL7。本文首先分析和鉴定出陆地棉中的HECT基因,并描绘它们的结构特征。试验获得GhUPL7的表达特征以及其上游序列在植物中的表达模式,检测和观察反义抑制转基因植株的胚珠、纤维和叶片表皮毛的变化,从而了解GhUPL7对棉花部分器官生长发育的影响,具体结果如下:1.陆地棉中的HECT基因进化分析根据HECT序列从陆地棉数据库检索出HECT蛋白序列在棉花中的25个同源基因,即HECT基因。25个陆地棉HECT基因与拟南芥UPLs进行进化树分析,可将GhUPLs归类为4个分支,即亚家族I,II,III,IV。其中Gh_A01G1811和Gh_D01G2049两个基因与克隆的GhUPL7基因相似度分别达到99%和98%,且基因Gh_A01G1811和Gh_D01G2049位于亚家族IV,与拟南芥AtUPL7亲缘关系最近。为更好地理解不同HECT基因的结构差异,根据它们的系统发生关系,分别将陆地棉基因组HECT蛋白结构域和HECT序列的外显子-内含子结构进行比较。结果显示:除C端的HECT结构域外,陆地棉HECT基因在N端包含了其他的结构域,推测这些结构域决定HECT蛋白与不同底物相互作用。HECT亚家族IV的N端没有结构域或存在未知结构域。关系密切的HECT基因在基因结构上也更相似,尤其是在外显子和内含子的数量上,主要的不同是它们各自的外显子-内含子长度。以上分析表明GhUPL7确实是HECT类泛素连接酶,并且在进化上具有保守性。2.GhUPL7的表达特征分析设计ghupl7特异性real-timepcr引物,检测ghupl7基因在不同器官的表达水平发现,其在柱头中表达量相对较高,而在其它器官中表达量很低。检测ghupl7基因在纤维不同时期的表达水平发现,纤维快速伸长期的表达量相对较高。3.ghupl7上游序列的克隆及分析克隆ghupl7上游2189bp序列,并运用植物启动子顺式作用元件数据库plantcare对ghupl7基因上游调控序列顺式作用元件数量和种类进行统计和汇总,结果显示存在激素反应相关元件,光反应相关的元件,组织特异性表达的元件、胁迫反应相关元件,结合位点和未知功能元件。可以看出这些顺式作用元件与植物生长发育密切相关。继而构建启动子表达载体,烟草遗传转化,在烟草器官gus染色结果显示:ghupl7启动子指导的gus基因主要在烟草柱头中表达,其次是花托、胚珠表面。进一步检测ghupl7启动子转基因烟草不同组织的gus活性:在柱头中酶活力最强,却仅有76.39nmol/min/mg,而不同时期的花托和子房相对较低,8dpa的花托和子房酶活力分别为11.13nmol/min/mg和10.28nmol/min/mg。以上结果表明ghupl7启动子指导的gus可能在花器官中表达。4.反义抑制ghupl7基因植株的种子增大,子指增加ghupl7是泛素化反应中一个特异性酶,而泛素化反应与植物生长发育息息相关。为探究该基因是否通过泛素化反应参与三大营养物质的代谢途径,构建植物表达载体camv35s::antisenseghupl7转入陆地棉(冀棉14)中,并通过设计rt引物进行real-timepcr筛选获得反义抑制ghupl7的转基因植株。收获繁殖继续筛选出t1代6个转化子共42株植株,对10dpa胚珠的总氮和可溶性总糖含量进行测定。结果显示:总氮含量最高增加26.60%;可溶性总糖含量最高增加32.96%。与之对应胚珠最后形成的种子增大,这与upl7拟南芥突变体表型相一致;产量性状统计发现:子指增加最高达14.86%。推测ghupl7可能参与泛素蛋白酶体途径,影响碳氮代谢。5.反义抑制ghupl7基因植株短绒增加,衣分无明显变化纤维的营养由胚珠供给,欲知反义抑制ghupl7对纤维发育的影响,对反义抑制ghupl7植株t1代进行纤维起始观察、糖含量测定、成熟纤维表型观察、产量性状统计。电镜观察反义抑制ghupl7基因对棉花纤维起始的影响,结果显示纤维细胞起始出现异常纤维,但纤维的突起数量却无明显变化。糖含量测定显示反义抑制ghupl7植株纤维可溶性总糖最高降低了30.66%;成熟纤维观察结果显示反义抑制ghupl7棉花长纤维不变或略变少,短绒增多。产量性状统计结果表明衣分变化不显著,最高降低了7.16%。6.反义抑制GhUPL7基因增加棉花叶片表皮毛的分支有研究报道UPLs家族的UPL3在拟南芥中与表皮毛的发育有关,故而对同为HECT家族的GhUPL7反义抑制植株叶片表皮毛进行观察,并对其分支数进行统计,结果显示1-3个分支的百分比明显减少,大于4个分支的百分比明显增多。该结果与upl7拟南芥突变体表皮毛分支数表型一致。反义抑制GhUPL7基因增加棉花叶片表皮毛的分支,推测表皮毛与纤维短绒发育具有相似机制。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S562

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