【作者】 李大红；

【导师】 梁建生；

【作者基本信息】 扬州大学 ， 植物学， 2008， 博士

【摘要】 RACK1蛋白属于一类含有WD-40重复结构蛋白家族的亚家族。WD-40重复蛋白是一类大的蛋白质家族,结构高度保守,广泛分布于真核生物和原核生物中,目前已鉴定到140多种蛋白属于WD-40重复蛋白家族。这类蛋白在多种生物生命活动过程中扮演非常重要的角色,如细胞信号转导、蛋白转运、细胞骨架的组装和解聚、RNA的加工、染色质的修饰及转录、细胞分裂、胞浆移动、细胞凋亡、光信号转导、细胞运动、开花、花序的发育以及分生组织的形成等。每个WD重复结构域包含40-60个氨基酸,WD结构域的N端一般是由Gly-His(GH)二肽组成的11到24个氨基酸残基,它的C端含有Trp-Asp(WD)二肽。最初RACK1蛋白被确定为蛋白激酶C(PKC)的受体,现在认为RACK1蛋白作为支架蛋白在多个信号转导途径中起重要作用。已有研究结果表明,拟南芥AtRACK1参与了植物对渗透胁迫信号的响应过程,并且与植物忍耐渗透胁迫密切相关。以AtRACK1蛋白的氨基酸序列为模板对水稻基因组序列进行比对,我们找到两个与编码AtRACK1蛋白高度同源的基因。在蛋白质水平两者与AtRACK1蛋白的同一性和相似性分别达到70%和80%。推测水稻的RACK1(OsRACK1)可能与拟南芥RACK1(AtRACK1)具有相似的功能。本研究以粳稻日本晴为研究材料,首先通过RT-PCR技术及T-A克隆法克隆了编码RACK1蛋白(NP-916988)的基因OsRACK1,并构建了OsRACK1基因的过表达载体和RNAi表达载体,再通过农杆菌介导的植物转化方法导入水稻并筛选出OsRACK1基因过表达和RNAi OsRACK1基因片段抑制表达的转基因植株,并进一步深入研究了OsRACK1基因在盐胁迫响应中的功能。主要结果如下:1提取水稻总RNA,并对其完整性进行了检测。2 RT法反转录合成了完整的OsRACK1基因的cDNA第一链,并以此为模板,用PCR技术扩增了双链OsRACK1的基因。序列分析表明,OsRACK1基因的大小为1199bp,包含了基因完整的编码序列,且与已发表的基因全序列完全相同,同源性达100%。3构建了带有由35S启动子控制的GUS基因的水稻OsRACK1基因的过表达载体和RNAi表达载体,并通过农杆菌介导的植物转化方法将其导入粳稻日本晴基因组中。4通过GUS染色进行初步的检测,然后对阳性植株进行基因组DNA PCR法鉴定,再用Southern blot对目的基因的真实性和拷贝数进行检测,最后通过RQ-PCR对OsRACK1基因的表达量进行鉴定获得了转基因水稻植株。5应用RACK1过表达(OX-2)和抑制表达(R-2)的转基因植株T1代种子进行的萌发试验及相关生理生化指标分析结果表明,在盐胁迫条件下OX-2种子萌发要快于对照和R-2种子的萌发;OX-2种子的根长、根数及芽长同样高于对照和R-2的;而且前者的可溶性糖的含量也较高,但可溶性蛋白和ABA含量低于对照与R-2的。表明RACK1过表达促进了盐胁迫条件下的种子萌发。6对相关转基因幼苗在盐胁迫条件下的生理分析结果表明,与对照(非转基因水稻)相比,在同样程度的盐胁迫条件下,RACK1过表达幼苗的相对含水量、叶绿素含量、脯氨酸含量、氧化酶的活性和ABA含量均低于对照和R-2幼苗,而MDA含量和电导率高于对照和R-2植株。由此推测OsRACK1蛋白对幼苗耐盐性具有负调节作用。7对OsRACK1基因在调节植株耐盐性生理与分子机理的进一步研究结果表明,OsRACK1过表达的转基因幼苗无论根还是叶的K/Na比均低于对照和OsRACK1表达受抑制的转基因幼苗,而OsRACK1表达受抑制的转基因幼苗根系对K的吸收和运输均大于对照和过表达转基因幼苗。在盐胁迫下,OsRACK1表达受抑制的转基因幼苗内源ABA的含量显著高于OsRACK1过表达的转基因幼苗和对照。与无盐胁迫处理相比,盐胁迫处理显著促进了DREB1和P5CS的表达,但各基因型间差异不明显。外源ABA处理显著促进了P5CS的表达,但对DREB1的表达无作用。RACK1基因的表达不受盐胁迫和外源ABA影响,在盐胁迫下转基因水稻内源ABA的含量与RACK1的表达量成负显著相关性。根据以上研究并结合前人的研究结果,推测OsRACK1对盐胁迫条件下种子萌发和幼苗生长的调节机制不同;OsRACK1对幼苗耐盐胁迫具有负调节作用,当OsRACK1基因表达受到抑制后,通过某种机制调节ABA合成,进而提高了水稻对盐胁迫的忍耐性。更多还原

【Abstract】 RACK1 belongs to a large family of WD40 repeat proteins and is a highly conserved protein found in both animals and plants.These proteins are characterized by the presence of repeats consisting of between 40 and 60 amino acids with two internal conserved dipeptide sequences,glycine-histidine（GH） and tryptophan-aspartic acid（WD）.Typically,WD40 repeat proteins contain 4 to 16 repeating regions consisting of a core of amino acids initiated by a GH dipeptide and ends with a WD dipeptide.The consensus sequence motif is {X6-94-[GH-X23-41-WD]}4-16.Because of their highly conserved structural motif,it was suggested that WD40 proteins play very diverse roles both in animals and in plants.RACK1 is now viewed as a versatile scaffold protein that can bind or act with numerous signalling molecules from diverse signal transduction pathways in a regulated fashion,including cAMP signalling,cell cycle control,Ca²⁺ release,ribosome assembly and mRNA translation regulation, cytoskeleton remodelling and proteasome degradation.RACK1 has been found to be a potential physical and functional linker between various signalling molecules.Recently,we have screened a mutant,Atrack1,from Arabidopsis thaliana that is highly tolerant to osmotic stress.Furthermore,we also found two highly homologous genes（NC008394 and NC008398） in the Oryza sativa to AtRACK1 from NCBI.The deduced amino acid sequences of these two genes-encoded proteins have 80%identity and 70%similarity to AtRACK1. However,at present,we know little about the functions of rice RACK1.Based on the results obtained from Arabidopsis,we deduced that OsRACK1 may also play important roles in the responses of rice to adverse stresses,especially to salt stress,drought stress,etc.To verify this hypothesis,we construct two kinds of transgenic rice plants,i.e.,OsRACK1 over-expression transgenic plant line and OsRACK1 inhibition transgenic plant line using RNA interference （RNAi） technique.The major results are as follows:1.We successfully constructed two kinds of transgenic rice plants by agrobacterium tumefaciens.The expression degrees of OsRACK1 gene were from 132%to 175%in over-expression transgenic plant lines and were 33%to 71%in OsRACK1 inhibition transgenic plant lines,respectively,as compared with that of non-transgenic rice plants.We chosed seeds harvested from T1 generation of one transgenic rice plant line from two kinds of two transgenic lines,respectively,in combination with those of non transgenic plants,as materials for the further analyses.2.The results from seed germination assay showed that,when exposed to salt conditions, seed germination of all genotypes was inhibited and the inhibition on seed germination was increased as the NaCl concentration increased.However,obvious differences in seed germination were observed among various genotypes,of which OsRACK1 over-expressing transgenic line was better than those of OsRACK1 inhibition transgenic plant line and non-transgenic rice seeds.Furthermore,the root length,root numbers and soluble suagr content of OsRACK1 over-expressing transgenic line were higher,but the contents of soluble protein and abseisic acid（ABA） were lower than those of OsRACK1 inhibition transgenic plant line and non-transgenic lines.These results indicated that OsRACK1 positively regulated seed germination.3.As compared to non-transgenic lines,the relative leaf water content,the contents of chlorophyll,proline and ABA,and the activities of anti-oxidases were lower in OsRACK1 over-expressing transgenic line than in OsRACK1 inhibition transgenic plant line under the same stress condition.Whereas the MDA content and the electric conductivity of OsRACK1 over-expressing transgenic line were higher than those of OsRACK1 inhibition transgenic plant line.4.Further studies on the possible physiological and molecular mechanisms of OsRACK1 gene in regulating rice salt-tolerance indicated that the K⁺/Na⁺ ratio both in roots and in leaves of OsRACK1 over-expressing transgenic line was lower than those of OsRACK1 inhibition transgenic plant line and non-transgenic lines.The abilities to absorb and transport K⁺ of OsRACK1 inhibition transgenic plant line were higher than those of OsRACK1 over-expressing transgenic line and non-transgenic lines.Salt stress significantly stimulated ABA biosynthesis and accumulation,however,this timulation was higher in OsRACK1 inhibition transgenic seedlings than in OsRACK1 over-expressing transgenic and non-transgenic seedlings.Real-time PCR analysis showed that salt stress significantly stimulated the expression of DREB1 and P5CS, as compared with that of non slat stress treatment,and no obvious differences were observed among various genotypes investigated.Exogenous ABA treatment had a stimulating effect on the expression of PSCS,but had no effect on the expression of DREB1.The expression of RACK1 was not affected by salt stress and exogenous ABA treatments.Under salt stress condition,the ABA content was negatively related to the RACK1 expression.According to the above results and other reports,it was suggested that the OsRACK1 gene exert its functions in salt-tolerance of seed germination and seedling growth with different mechanisms.OsRACK1 negatively regulated salt-tolerance of seedling growth largely through controlling ABA biosynthesis.That is,when the expression of OsRACK1 was inhibited,the ABA biosynthesis was stimulated by some unknown mechanisms,as a consequence,the tolerance to salt stress was ehnhanced.Of course,the detailed physiological and molecular mechanisms of OsRACK1 action need to be further investigation.更多还原