拟南芥PMRP的表达特性及功能分析

doi:10.3864/j.issn.0578-1752.2014.15.019

摘要/Abstract

摘要： 【目的】分析拟南芥中功能未知基因PMRP（putative membrane related protein）的表达特性；明确PMRP在调控拟南芥生长发育过程中的作用。【方法】利用生物信息学方法，寻找拟南芥中与PMRP含相同结构域的基因，并绘制进化树；利用Real-time PCR技术分析PMRP在生长8和21 d的拟南芥根、茎中的相对表达量，比较生长21 d的拟南芥第1、2、3、4对莲座叶及茎生叶中PMRP的表达情况，分析花器官的萼片、花瓣和雄蕊及生长后期拟南芥种子中PMRP的表达量的高低；构建35S::PMRP过表达载体，转化Columbia野生型拟南芥，通过RT-PCR技术验证PMRP的表达量，获得过表达PMRP的转基因植株；通过对过表达PMRP的转基因拟南芥的表型观察，分析PMRP对拟南芥叶片发生、茎的生长部位等的调控作用；通过对过表达PMRP转基因拟南芥茎秆横切面的石蜡切片观察，分析PMRP在茎秆维管束木质部和韧皮部分化过程的作用；通过对花器官的解剖学观察，明确PMRP对拟南芥花器官生长发育的影响；通过对过表达PMRP转基因拟南芥果荚形成的观察，分析PMRP对拟南芥育性的影响。【结果】PMRP是一个含有START保守域的411个氨基酸的蛋白质，具有跨膜结构域，在拟南芥中含有START结构的基因共35个；Real-time PCR结果表明，PMRP在茎生叶中表达量最高（相对表达量约为2 935）、莲座叶中次之，且莲座叶生长时间越长，PMRP的表达量越多（PMRP在第1、2、3、4对莲座叶中的相对表达量分别约为1 650、1 113、734、507），然后是花器官中的萼片（PMRP相对表达量约为937），PMRP在茎、根、种子中都有的表达，但相对表达量都低于270；PMRP在花器官中雄蕊的相对表达量最低（约为64），远远低于同属花器官的萼片中PMRP表达量（937），PMRP在根、茎中的表达量随生长时间的增加而增加，PMRP在8和21 d的根中相对表达量分别为154和222，PMRP在8和21 d的茎中相对表达量分别为200和264；过表达PMRP转基因拟南芥表现为在枝条上产生莲座叶，茎秆容易倒伏，维管束没有明显的形成层，且木质部和韧皮部排列紊乱，花器官中雄蕊的花丝变短，形成的角果数量减少，育性降低。【结论】START结构域在功能上极度保守；PMRP在拟南芥不同组织器官均有表达，且随着时间的延长，PMRP的表达量也增加，但在花器官的雄蕊表达最低，一旦过表达PMRP，拟南芥花器官的雄蕊发育异常，造成育性降低；PMRP对拟南芥的叶片发生、茎秆维管束分化和花器官发育具有重要作用。

关键词: 拟南芥 , PMRP , 功能分析

Abstract: 【Objective】The objective of this study is to analyze the expression characteristics of PMRP (putative membrane related protein)gene (the biological funcion was unknown )in Arabidopsis, and make clear of the regulation function of PMRP in Arabidopsis development process.【Method】Bioinformatics method was used to look for the genes which contain the same domain with PMRP in Arabidopsis and draw the phylogenetic tree. Real-time PCR technique was used to analyze the expression level of PMRP in root and stem tissues of 8 and 21 days Arabidopsis, compare the expression of PMRP gene in the 1st, 2nd, 3rd and the 4th pairs of rosette leaves and cauline leaves in 21 days Arabidopsis, analyze the expression level of PMRP in flower organs (such as sepals, stamens and petals) and seeds of Arabidopsis. The gain-of-function mutant of PMRP were obtained by constructing a vector of PMRP gene driven by the 35S promoter of the cauliflower mosaic virus, and introduced it into wild type Arabidopsis Columbia, then tested the expression level of PMRP by RT-PCR technique. The regulating effect of PMRP gene on the growth sites of leaves and stems was analyzed by observing the phenotype of the 35S:PMRP transgenic plants. The function of PMRP gene in differentiation of vascular xylem and phloem was observed using the paraffin sections of the stem transverse section in the PMRP over-expressing transgenic Arabidopsis. The effect of PMRP on the growth and development of Arabidopsis floral organ was studied through observation of the floral organ anatomy. The effect of PMRP on fertility in Arabidopsis thaliana was analyzed by observing the pod formation in the PMRP over-expressing transgenic Arabidopsis. 【Result】PMRP is a 411 amino acids protein which containing a START domain and have transmenbrane segments, there are 35 START proteins in Arabidopsis. Real-time PCR analysis results showed that the expression level of PMRP was higher in cauline leaves ( the relative expression of PMRP was about 2 935) and then the rosette leaves, the growth time of rosette leaves was longer, the more expression of PMRP was tested (the relative expression of PMRP in the 1st, 2nd, 3rd and 4th pairs of the rosette leaves were 1 650, 1 113, 734, and 507, respectively) , then the sepals floral tissues (PMRP relative expression quantity is about 937), PMRP had distribution in the stem, root and seed , but the relative expression amount was less than 270. The relative expression level of PMRP in stamens was the lowest (about 64), far below the expression quantity of PMRP in sepals (937). The expression level of PMRP gene was increased with the increase of growth time, the relative expression of PMRP were 154 and 222, respectively, in 8 and 21 d roots, and 200 and 264, respectively, in 8 and 21 d stems. The rosette leaves were emerged from the branches, stalk lodging susceptible, vascular had no obvious cambium and the xylem and phloem arranged in disorder, the filaments became shorter in floral organs, the number of pod decreased, and the fertility reduced.【Conclusion】The START domain is extremely conservative in function. PMRP was expressed in different organs of A. thaliana, with the extension of time, the expression level of PMRP was also increased. The level of PMRP in the flower stamens was the lowest, once PMRP was over-expressed in Arabidopsis floral organs, stamens dysplasia, resulting in reduced fertility. PMRP had important function in regulating the origination of leaves, the vascular differentiation and development of floral organs.

Key words: Arabidopsis , PMRP , function analysis

张昊, 由诗东, 高静, 张海丽, 李生辉, 邢继红, 王凤茹, 董金皋. 拟南芥PMRP的表达特性及功能分析[J]. 中国农业科学, 2014, 47(15): 3094-3102.

ZHANG Hao, YOU Shi-Dong, GAO Jing, ZHANG Hai-Li, LI Sheng-Hui, XING Ji-Hong, WANG Feng-Ru, DONG Jin-Gao. PMRP Expression Characteristics and Analysis of the Function in Arabidopsis[J]. Scientia Agricultura Sinica, 2014, 47(15): 3094-3102.

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