梭梭14-3-3蛋白基因HaFT-5～9的克隆分析及HaFT-4功能验证

发布时间：2018-06-12 22:56

本文选题：梭梭 + 14-3-3蛋白基因　；参考：《新疆农业大学》2016年硕士论文

【摘要】：14-3-3蛋白是以同源或者异源二聚体形式存在的一类保守性很高、可以多基因编码并且具有调节功能的蛋白质家族。在调节转录因子的核定位、调控基因的表达和酶活性的调控等多种细胞活动中起到重要作用。本论文以梭梭为研究对象,采用RT-PCR方法分离并成功克隆出5个14-3-3蛋白基因,进行了生物信息学的初步分析。采用q RT-PCR方法研究5个基因在干旱胁迫下表达水平。利用农杆菌介导转化法获得了HaFT-4基因转烟草植株,并对转HaFT-4基因烟草进行相关分析。主要研究结果如下:1.成功克隆5个梭梭14-3-3蛋白基因,命名为HaFT-5、Ha FT-6、HaFT-7、HaFT-8和Ha FT-9。5个14-3-3蛋白基因ORF大小分别为420 bp、681 bp、789 bp、792 bp和789 bp分别编码139、226、262、263和262个氨基酸。分别对这5个基因编码的氨基酸序列的生物信息学进行了分析,预测得出,HaFT-5为稳定蛋白,其余4个蛋白为不稳定蛋白。这5个蛋白都是亲水蛋白,均没有信号肽,同时不存在跨膜结构。蛋白质序列相似性比对发现,5个14-3-3蛋白中HaFT-5与Ha FT-6与甜菜的同源性最高,HaFT-7~HaFT-9与菠菜的同源性最高,均达到90%以上。分子进化树可以得出,HaFT-5与甜菜聚为一支,HaFT-6与葡萄和百合聚为一支,HaFT-7与Ha FT-9被聚为一支的同时均和菠菜聚为一支,HaFT-8与菠菜聚为一支。2.利用实时荧光定量对5个14-3-3蛋白基因在20%PEG6000模拟干旱胁迫处理0 h、4 h、8 h、12 h和24 h的表达量进行分析,HaFT-5基因在12 h产生最高表达量,后明显下降。Ha FT-6~Ha FT-9基因均在干旱胁迫8 h有最大表达量,之后逐渐降低。HaFT-9基因的相对表达量有最显著的变化,但是Ha FT-6基因与未胁迫时相比表达量变化不显著。3.成功构建了pCAMBIA1304-HaFT-4植物表达载体,用农杆菌介导转化叶盘法将该基因转入烟草中,获得5株转基因烟草植株。选择3株转基因植株及野生型烟草植株叶片进行干旱、盐及低温胁迫后分析丙二醛、脯氨酸、可溶性糖和叶绿素的变化情况。结果表明,3株转HaFT-4基因植株经过胁迫后变化趋势较为相似,但是3株转HaFT-4基因烟草植株相较于野生型烟草植株的丙二醛含量降低,脯氨酸积累增加,可溶性糖含量升高,叶绿素含量也升高。综合来看,转Ha FT-4基因烟草对于逆境具有更强的抵御能力。
[Abstract]:14-3-3 proteins are a family of proteins with high conserved status in the form of homologous or heterodimer, which can be encoded by multiple genes and have regulatory functions. It plays an important role in regulating the nuclear localization of transcription factors, gene expression and enzyme activity. In this paper, five 14-3-3 protein genes were isolated and cloned successfully by RT-PCR, and the preliminary bioinformatics analysis was carried out. The expression level of five genes under drought stress was studied by Q RT-PCR. HaFT-4 transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transformation. The main results are as follows: 1. Five 14-3-3 protein genes of Haloxylon ammodendron were successfully cloned and named HaFT-5 Ha FT-6 HaFT-7 HaFT-8 and Ha FT-9.5 14-3-3 protein genes. The ORF sizes of these genes were 420bpn681 BP, 789 BP, 792 BP and 789 BP, respectively, encoding 1392262263 and 262 amino acids, respectively. The bioinformatics analysis of the amino acid sequences encoded by these five genes showed that HaFT-5 was a stable protein and the other four proteins were unstable proteins. All the five proteins were hydrophilic, without signal peptide and no transmembrane structure. The homology of HaFT-5 and Ha FT-6 with beet was the highest among the five 14-3-3 proteins. The homology of HaFT-7 and HaFT-9 with spinach was the highest, and the homology of HaFT-5 and Ha FT-6 was over 90%. The molecular phylogenetic tree showed that HaFT-5 and beet formed a single HaFT-6 with grape and lily, HaFT-7 and Ha FT-9 were clustered into one, while spinach and spinach were clustered into a branch of HaFT-8 and spinach into one branch of .2.The molecular evolution tree showed that HaFT-5 and sugar beet formed a branch of HaFT-6, and that of grape and lily were one branch of HaFT-7 and Ha FT-9. The expression of 5 14-3-3 protein genes in simulated drought stress of 20 PEG6000 was analyzed by real-time fluorescence quantitative analysis. The highest expression of HaFT-5 gene was observed at 12 h after treatment of 0 h, 4 h and 8 h, and 24 h, respectively. The relative expression of HaFT-9 gene decreased significantly after drought stress for 8 h, but Ha FT-6 gene had no significant change compared with that under drought stress, but the relative expression level of Ha FT-6 gene was not significantly changed compared with that of non-stress (P < 0.05), but the relative expression level of HaFT-9 gene decreased significantly after drought stress for 8 h, but the change of Ha FT-6 gene was not significant compared with that under drought stress. The plant expression vector pCAMBIA1304-HaFT-4 was successfully constructed and transformed into tobacco by Agrobacterium tumefaciens. The changes of malondialdehyde (MDA), proline, soluble sugar and chlorophyll in leaves of 3 transgenic and wild-type tobacco plants were analyzed after drought, salt and low temperature stress. The results showed that the changes of malondialdehyde (MDA) content, proline accumulation and soluble sugar content in three transgenic HaFT-4 transgenic tobacco plants were lower than those in wild type tobacco plants, but the change trend of HaFT-4 transgenic plants was similar to that of wild type tobacco plants, but the content of soluble sugar in three transgenic HaFT-4 transgenic tobacco plants was higher than that in wild type tobacco plants. Chlorophyll content also increased. In general, transgenic tobacco with Ha FT-4 gene had stronger resistance to stress.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.99;Q943.2

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