水稻叶片衰老相关蛋白磷酸酶编码基因OsSAPP2的克隆及初步功能分析

发布时间：2018-03-26 06:00

  本文选题：水稻　切入点：叶片衰老　出处：《沈阳农业大学》2017年硕士论文

【摘要】：叶片衰老是叶片生长发育的最后一个阶段,研究叶片衰老对整株植物而言具有重要意义。蛋白磷酸酶催化的蛋白质去磷酸化在植物多种生命过程中有重要地位,其中2C型蛋白磷酸酶(PP2C)是植物中数量较多的一类,但关于PP2C参与水稻叶片衰老进程调控的报道还比较少。因此,关于参与水稻叶片衰老过程中蛋白磷酸酶基因的鉴定、克隆及功能分析具有重要意义。1、水稻参与叶片衰老调控的PP2C候选基因的筛选在前期实验中,通过公共Microarray芯片数据分析、生物信息学计算结合序列同源性比对,筛选到7个可能参与水稻叶片衰老进程调控的PP2C基因,命名OsS4PP1-7.在此基础上利用荧光定量RT-PCR检测OsSAPP1-7在人工黑暗诱导水稻衰老过程中的表达情况,发现OsSAPPl、OsSAPP2、OsAPP3、OsSAPP5和OsSAPP7变化较显著,随后在自然衰老中发现OsS4PP2、OsS4PP3表达变化最强烈,因此将OsSAPP2、OsSAPP3作为后续研究的目标基因。2、OsS4PP2基因的克隆与植物双元表达载体构建构建了过表达植物双元表达载体(35S-OsSAPP2、35S-OsSAPP3)、启动子-GUS报告基因植物双元表达载体(ProossAPP2-GUS、ProossAP3-GUS),利用农杆菌介导的遗传转化法对拟南芥和水稻进行了遗传转化,通过卡那霉素或潮霉素抗性筛选、GUS组织化学染色鉴定、PCR检测、荧光定量RT-PCR检测,获得相应的转基因株系。3、35S-OsSAPP3转基因植物的获得实验中无法获得35S-OsSAPP3转基因水稻阳性植株,同样组成型启动子过表达OsS4PP3基因导致转基因拟南芥早期衰亡无法完成生活史,推测OsSAPP3在组成型启动子驱动下过表达会导致植物死亡。因此将组成型启动子替换成可诱导型启动子,重新构建植物双元表达载体、获得转基因植物后再进行分析。4、35S-OsSAPP2转基因植物的表型观察35S-OsSAPP2转基因水稻和拟南芥叶片褪色较快,萎蔫衰老情况加剧,叶绿素含量下降速率高于对照。35S-OsSAPP2转基因拟南芥抽苔和开花时间较早,莲座叶小且少,株高较高,种子千粒重较小和籽粒长宽比较大。另外,在黑暗诱导衰老下,35S-OsSAPP2转基因拟南芥叶片黄化严重、叶绿素含量下降幅度大。以上结果表明OsSAPP2过表达会引起转基因植物早衰。5、35S-OsSAPP2转基因植物的基因表达分析水稻衰老标志基因OsSM-1和OsSM-2在35S-OsSAPP2转基因水稻叶片中的表达量均上升,而OsTZF1的表达量下降;35S-OsSAPP2转基因拟南芥叶片衰老相关基因(S4G2)、衰老关键转录因子(NAC1NAC2、AN4CP、WRKY6)和叶绿素降解关键酶编码基因(ACD1)的表达量较野生型高且上升快;光合作用相关基因(RbcL、RbcS)的表达量大幅度下降,在人工黑暗诱导衰老下也观察到了类似结果。6、35S-OsSAPP2转基因植物对植物激素的响应植物衰老促进激素乙烯和脱落酸会加速35S-OsSAPP2转基因水稻的离体叶片早衰,而植物衰老抑制激素生长素可以有效缓解由OsSAPP2基因过表达造成的水稻叶片早衰。综上所述,本研究发现过表达OsS4PP2转基因植物具有早衰表型,因此认为OsSAPP2基因是水稻叶片衰老的正向调节因子。
[Abstract]:Leaf senescence is the last stage of leaf growth, leaf senescence research has important significance for the whole plant. Protein phosphatase catalytic protein phosphorylation plays an important role in many biological processes in plants, including type 2C protein phosphatase (PP2C) is a kind of large number of plants, but the involvement of PP2C in rice regulation of leaf senescence was rarely reported. Therefore, the identification of protein phosphatase involved during leaf senescence in rice gene, cloning and functional analysis of.1 has an important significance, screening the candidate gene of PP2C in rice leaf senescence regulation in the previous experiment, through the public Microarray microarray data analysis, bioinformatics computing with sequence homology comparison on screening to 7 may be involved in rice leaf senescence regulation of PP2C gene, named OsS4PP1-7. on the basis of the use of fluorescent quantitative RT-P CR OsSAPP1-7 was detected in dark induced expression of artificial aging process in rice, OsSAPPl, OsSAPP2, OsAPP3, OsSAPP5 and OsSAPP7 change significantly, then found OsS4PP2 in natural senescence, the expression of OsS4PP3 is the strongest, so OsSAPP2, OsSAPP3 as the following target genes of.2, OsS4PP2 gene cloning and plant expression vector construction of over expression of plant binary expression vector (35S-OsSAPP2,35S-OsSAPP3), binary expression vector of -GUS promoter reporter gene plants (ProossAPP2-GUS, ProossAP3-GUS), using Agrobacterium mediated genetic transformation method for genetic transformation of Arabidopsis and rice by kanamycin or hygromycin resistance screening, GUS histochemical staining, PCR assay, fluorescence quantitative RT-PCR detection, to obtain the corresponding transgenic lines of.3,35S-OsSAPP3 transgenic plants was not obtained in the experiment 35S-O SSAPP3 transgenic rice plants were the same, the constitutive promoter of OsS4PP3 gene in transgenic Arabidopsis early decline to complete life history expression, suggesting that OsSAPP3 in the drive constitutive promoter over expression may lead to the death of plants. So the constitutive promoter replaced inducible promoter, construction of plant binary expression vector observation 35S-OsSAPP2 transgenic rice and Arabidopsis leaves fade faster phenotype of.4,35S-OsSAPP2 transgenic plants were obtained after analysis of transgenic plants, increased the chlorophyll content decreased wilting and aging, the rate is higher than that of the control.35S-OsSAPP2 transgenic Arabidopsis bolting and flowering time earlier, rosette Ye Xiaoqie, higher plant height, smaller seed weight and grain length width is relatively large. In addition, in the dark induced senescence, 35S-OsSAPP2 transgenic Arabidopsis leaves chlorophyll content decreased seriously. Large amplitude. The above results indicated that overexpression of OsSAPP2 can cause analysis of rice senescence marker gene expression of OsSM-1 and OsSM-2 in 35S-OsSAPP2 transgenic rice leaves were significantly increased the expression of transgenic plant senescence.5,35S-OsSAPP2 transgenic plants gene, whereas the expression of OsTZF1 decreased; 35S-OsSAPP2 transgenic Arabidopsis leaf senescence associated genes (S4G2), a key transcription factor of aging (NAC1NAC2, AN4CP, WRKY6) and encoding key enzymes of chlorophyll degradation gene (ACD1) expression and increased faster than the wild type; photosynthesis related genes (RbcL, RbcS) the expression volume decreased significantly, in the artificial dark induced senescence was also observed under similar results of.6,35S-OsSAPP2 transgenic plants on plant hormone response promoting plant senescence hormone ethylene and abscisic acid can accelerate 35S-OsSAPP2 transgenic rice leaves in vitro senescence, and inhibit growth hormone and plant senescence It can effectively alleviate premature senescence of rice leaves caused by overexpression of OsSAPP2 gene. In conclusion, this study found that overexpressing OsS4PP2 transgenic plants had premature decay phenotype, so OsSAPP2 gene is a positive regulator of leaf senescence in rice.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
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本文编号：1666640