【作者】 李鹏；

【导师】 郭旺珍；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2016， 博士

【摘要】 棉花作为世界上最重要的经济作物之一,给人类提供了非常重要的天然纤维。随着棉花基因组序列解析的巨大进展,从棉花全基因组中发掘重要的纤维发育的优良基因并功能分析,将促进棉花的品质和产量改良。Rab蛋白是小G蛋白家族中最大的一个家族,其作为棉花中重要的一个家族,很多成员在纤维发育过程中都发挥十分重要的作用。通过全基因组分析Rab基因家族成员的序列、分类、基本生物信息、基因结构、染色体分布以及表达特征,可为相关基因的克隆和功能研究提供重要依据。到目前为止,拟南芥57个Rab基因家族成员的鉴定工作已完成,但在棉花中,还未有相关基因家族的研究报道。本研究结合释放的棉花基因组信息,对棉花Rab基因家族成员进行分析。进一步通过差异表达分析,发现一个在纤维伸长发育中差异表达的Rab基因,结合其在家族表达模式热图,发现其在纤维伸长期中优势表达,将该基因命名为GhRab11d3。对该基因及其启动子克隆,并对其特征和功能进行了系统研究,主要研究结果如下:通过检索二倍体雷蒙德氏棉的全基因组数据库,一共获得87个属于Rab基因家族的成员,通过拟南芥的命名法则并结合哺乳动物的命名规则,对每个成员分别进行命名。生物信息分析表明,棉花Rab基因家族成员氨基酸数目是200-300 aa,蛋白分子量都处于20-30Kda之间,这与Rab蛋白本身所具有的性质保持一致。染色体分布图表明除2个基因没有找到相对应的物理位置外,其余85个Rab基因家族成员分布在雷蒙德氏棉的全部13条染色体上,在第10条染色体上分布的Rab基因最多,有11个,而在第4条染色体上分布的基因最少,只有2个。系统进化分析表明,这87个成员被分为八大类,与已报道的拟南芥Rab家族分类相对应,每一大类成员之间有着相似的功能。基因结构分析发现,同一大类成员中的内含子数目基本一致,即同一大类成员的基因结构基本相似。对雷蒙德氏棉中所对应的TM-1成员中其转录组数据的表达模式分析,发现大约有161个基因至少在一个组织器官中被检测到有表达,说明大多数Rab家族成员在棉花的不同生长发育阶段都至少会发挥一定功能。大约有一半基因会在棉花的营养器官、花器官以及纤维胚珠器官中都有所表达,说明这些基因是组成型表达基因,它们会参与棉花的整个生长发育进程,在功能上具有多样性。少数基因分别在某个或某类组织器官中表达,说明这些基因在功能上比较专一。还有一些基因的表达模式则不明确,未发现规律。Li1野生型具有正常发育的纤维,而突变体具有超短纤维表型。通过差异显示分析,发现了一个在Li1突变体和野生型纤维发育时期差异表达的Rab基因。进一步对该基因在Li1突变体和野生型不同纤维发育组织中进行定量表达分析,证明在8DPA、10DPA、12DPA以及15DPA的纤维组织中,该基因在野生型中的表达量极显著高于突变体,表明该基因对纤维伸长具有正向调控作用。结合Rab基因家族研究结果,发现该基因对应命名为GhRab11d3,从表达模式热图结果可以看出该基因属于组成型表达,但是在纤维伸长期优势表达。GhRab11d3基因全长cDNA为690 bp,编码229 aa,具有Rab蛋白典型的四个鸟嘌呤核苷酸结合结构域以及C末端的两个半胱氨酸的结构特点,等电点pI为6.84,分子量Mw大小为25.16 KDa。该基因在陆地棉遗传标准系TM-1的各个组织器官中均表达,尤其在纤维快速伸长期的表达量最高。通过转化棉花原生质体以及注射烟草的体内体外亚细胞定位方法,结果表明该蛋白位于细胞的内膜系统中,包括细胞膜、细胞质以及细胞核,通过细胞器Marker蛋白标记的红色荧光信号与目的蛋白的绿色荧光信号进行共定位,发现涉及到的具体细胞器有内质网。为了进一步验证该基因的功能,我们构建了组成型强启动子35S和纤维特异启动子RDL驱动的GhRab11d3的正反义表达两套载体,通过农杆菌介导进行了棉花的稳定遗传转化研究。除了 35S-ASC载体在侵染50天后出现愈伤褐化死亡的现象,无法获得转基因后代株系以外,其余三个载体均获得了一批转基因成功的后代纯合株系。通过转基因后代与受体W0在纤维伸长期5DPA、10DPA、15DPA的定量qPCR分析,发现35S-SC的303株系和RDL-SC的210株系等其目标基因表达量显著增加,而RDL-ASC的98株系其目标基因表达量被显著抑制,因此选择这三个株系开展目标基因功能验证分析。通过比较这三个转基因株系与受体W0在纤维发育10DPA、15DPA、20DPA和成熟期的纤维长度,发现正义载体的纤维显著变长,反义载体的纤维长度显著缩短。其10DPA和15DPA铃的大小分析表明,相比受体W0,转基因正义载体的铃变大,反义载体的铃变小。通过酵母双杂交共转化以及双分子荧光互补BiFC分析,证明GhRab11d3与在纤维伸长期优势表达的四个细胞骨架蛋白GhACTa、GhACTb、GhACT2、GhACT4均存在互作,且与GhACTb和GhACT2的互作更强。暗示GhRab11d3在囊泡运输过程中会沿着细胞骨架蛋白ACTIN运输细胞壁的组份多糖类物质,在此过程中,一方面会对细胞骨架蛋白微丝F-Actin的形态结构产生一定影响,另一方面会对细胞骨架蛋白微丝F-Actin的密度或者数量产生变化,同时,纤维细胞顶端的囊泡数量产生变化,使得囊泡运输能力受到影响,最终运输到细胞壁的果胶物质含量也会受到影响。我们发现正义载体的纤维组织中果胶含量显著增加,而反义载体的果胶含量减少,这可能是使纤维长度发生变化的主要原因。为了更好地认识该基因的功能,克隆了位于GhRab11d3起始密码子ATG上游1,546 bp长度的序列,以此作为该基因的启动子,并将其命名为pGhRab11d3进行启动子特征分析。预测该启动子转录起始位点TSS位于ATG上游312bp处的碱基T处,将其标记为-312 bp。通过PlantCARE和PLACE在线网站对启动子的顺式作用元件进行预测,发现了 TATA-box和CAAT-box基本元件,重要转录因子结合位点元件,几种激素诱导元件以及调控种子萌发和花粉管生长的作用元件等等。将pBI121载体用HindⅢ和BamHI酶切位点切掉35S启动子后,构建五段连有GUUS报告基因的缺失启动子载体,分别为 P1(196bp)、P2(385bp)、P3(546bp)、P4(746bp)以及P5(1,546bp)。我们对每段缺失启动子载体进行了烟草的稳定遗传转化,在转基因烟草中,pGhRab11d3主要在烟草的花瓣、花药、花梗以及胚珠这些生殖器官中发挥功能。GUS染色和GUS酶活测定结果表明,P1(196 bp)和P2(385 bp)与对照WT没有明显差别,从P3(546 bp)开始,可以观察到明显的GUS染色,GUS酶活也是极显著增加,说明该启动子从P3(546 bp)开始具有转录活性,所以认为起始密码子ATG上游的546 bp启动子长度对于驱使下游GUS基因的表达是所必需的。从中还可以推断出pGhRab11d3的核心启动子元件位于P2(385 bp)和P3(546 bp)之间,可能是MYB转录因子结合位点(492 bp)或者MADS转录因子结合位点(417bp)。另外,我们发现546 bp的上游序列中还含有增强下游基因表达的调控元件。GhRab11d3的启动子中可以被三种激素(包括GA、IAA以及6-BA)所诱导,但是并不能被ABA所诱导。通过将构建好的五个缺失启动子载体质粒进行基因枪轰击棉花胚珠,并对培养的胚珠长出纤维以后进行GUS染色,发现P3、P4和P5载体的纤维上面均有一定程度的GUS染色,从而再次证明GhRab11d3在纤维发育过程中发挥一定功能。更多还原

【Abstract】 As one of the most important economic crops in the world,cotton provides a very important natural fiber for human beings.With the great progress of cotton genome sequence analysis,we tried to find the important genes related with fiber development in the whole genome of cotton and analysis function,and it will also improve the quality and yield of cotton.Rab protein family is the largest subfamily of small G protein family.As one of the most important families in cotton,the Rab gene family plays a very important role in the process of fiber development.We can gain an important basis for the cloning and functional analysis of related genes by whole genome analysis about sequence,classification,basicbiological information,gene structure,genome distribution and expression characteristics of Rab gene family members.So far,the identification of 57 members of the Rab gene family in Arabidopsis has been completed,but in cotton,the relevant gene family has never been reported.In this study,combined with the release information of cotton genome,the cotton Rab gene family members were analyzed.Further by differential expression analysis,we found a Rab gene which expressed different in fiber elongation.Combined with its expression pattern in the family,we found it was dominantly expressed in the fiber elongation stage.The gene was named GhRab11d3.We cloned this gene and its promoter,and studyed its characteristics and functions systematically.The main results are as follows:We got a total of 87 members of the Rab gene family by retrieving the whole genome database of diploid Gossypium raimondii.Through the naming rules of Arabidopsis and the naming rules of mammals,each member was named respectively.Biological information analysis showed that the number of amino acid is 200 to 300 aa among Rab gene family members in cotton and the protein molecular weight is between 20 Kda and 30Kda,which is consistent with the nature of the Rab protein itself.Chromosome distribution map showed that 2 genes were not found in corresponding physical location,and the remaining 85 Rab protein gene family members are distributed in all 13 chromosomes of Gossypium raimondii.The distribution of Rab gene on chromosome 10 was the most,there were 11 and only 2 genes were distributed on chromosome 4.Phylogenetic analysis showed that the 87 members were divided into eight categories,which correspond to the reported Rab family of Arabidopsis thaliana.Members of each class have a similar function.After analyzing the genetic structure of all the members,we found that the number of introns in the same class was basically the same,that is,the genetic structure of the same class members was basically similar.After analyzing the transcriptome data expression pattern of TM-1 members of corresponding Gossypium raimondii,it is found that about 161 genes were detected expression at least in a tissue and organ.It shows that most of the Rab family members will perform certain functionin in different growth and development stages.About half of the genes were expressed in the vegetative organs,flower organs,fiber and ovuleorgans of cotton,indicating that these genes are constitutively expressed genes and they will participate in the growing developmental process and have diversity in function.Only a small number of genes are expressed in one or some kind of tissues,indicating that these genes are functionally specific.The expression pattern of some genes is not clear enough and we have no found regularity.Li1 wild type has the normal developmental fiber,and the mutant has an ultra short fiber phenotype.Differential expression of a Rab gene in fiber development stages of Li 1 mutant and wild type was found by differential display analysis.Through the quantitative expression analysis of this gene in different fiber development tissues of Lil mutant and wild type,it was proved that the expression of 8DPA,10DPA,12DPA and 15DPA was significantly higher in the wild type than that in the mutant.It was proved that the gene has a positive regulatory effect on fiber elongation.Combined with the analysis results of Rab gene family,it was found that the gene was named as GhRab11d3.From the expression pattern,we can see that the gene belongs to constitutive expression,and it is dominantly expressed in the fiber elongation stage.It has 690 bp full-length cDNA of GhRablld3,encodes a protein of 229,with typical of four guanine nucleotide binding domain and two cysteine structure of C terminus,and pI was 6.84,molecular weight Mw 25.16KDa.This gene has a certain level of expression in all tissues and organs of TM-1,especially in the rapidlongation period of fiber.Subcellular localization was performed by means of the method of transformation of cotton protoplasts and injection of tobacco.The results showed that the protein was located in the cell membrane system,including the cell membrane,cytoplasm and nucleus.The red fluorescent signal of marker protein was colocated with the target protein,and it was found that there was a specific organelle involved in the endoplasmic reticulum.In order to further verify the function of this gene,the sense and antisense constructs of GhRablld3 driven by the constitutive promoter 35S and the fiber specific promoter RDL respectively were carried out through the genetic transformation of cotton.In addition to callus browning phenomenon of the 35S-ASC vector in the 50 days after the infection,can not get the transgenic progeny plants,the remaining three vectors were obtained a number of transgenic homozygous lines.Through transgenic progeny and receptor WO in the fiber elongation stage 5dpa,lOdpa and 15dpa qPCR analysis,we found expression of target gene in 35S-SC 303 line and RDL-SC 210 line was significantly increased,expression of target gene in RDL-ASC 98 line was significantly inhibited,so the choice of the three lines was performed to verify function.In comparison with the fiber length of three transgenic lines and receptor WO in 10DPA,15DPA,20DPA and mature stage of fiber development,it was found that the fiber length of the sense construct was significantly longer and the fiber length of the antisense construct was significantly shorted.Analysis of the size of the boll in 10 DPA and 15 DPA showed that,compared with the receptor W0,the boll of the sense vector of the transgenic plants became larger,and the boll of the antisense vector became smaller.Analysis of cotransformation of yeast two hybrid and bimolecular fluorescence complementation(BiFC)demonstrated that GhRablld3 interacted with four cell skeleton protein ACTa,ACTb,ACT2,ACT4 which are dominantly expressed in fiber elongation stage,and interacted stronger with ACTb and ACT2.This finding reveals that GhRab11d3 transported a component of the cell wall in the vesicle transport process along the cytoskeleton protein ACTIN.In the course of this process,it has some influence on the morphological structure of cytoskeletal protein actin filament F-Actin,and produces changes in the density or number of cytoskeletal protein actin filaments F-Actin.At the same time,the number of vesicles at the top of the fiber cell is changed,so that the transport capacity of the vesicles is affected.The pectin content of the final shipping to the cell wall will also be affected.We found that the pectin content of sense construct in fiber tissues was significantly increased,and the antisense vector was decreased,which may be the main reason for the change of fiber length.To better understand the function of this gene,we cloned a 1,546 bp upstream sequence of the GhRablld3 initiation codon ATG,which was used as the promoter of the gene and named it pGhRablld3 and then did promoter characteristic analysis.Prediction of the promoter transcription start site TSS is locatedthe base Tin the ATG upstream 312bp,which is marked as-312 bp.By plantCARE and PLACE online website of predicting promoter cis acting elements,wefound that TATA-box and CAAT-box,important transcription factor binding site elements,several hormone induced elements and the acting elements of regulating seed germination and pollen tube growth.The vector pBI121 with HindⅢ and BamHl cut 35S promoter.We constructed five segments deletion promoter vectorconnected with GUS reporter gene,respectively P1(196 bp),P2(385 bp),P3(546 bp),P4(746 bp)and P5(1,546 bp).We have carried out the stable genetic transformation of tobacco with each deletion promoter.In transgenic tobacco,pGhRab 11 d3 is mainly in the reproductive organs of tobacco,such as petals,anthers,peduncles and ovules.GUS staining and GUS enzyme activity assay showed that P1(196 bp)and P2(385 bp)were not significantly different from WT.From P3(546 bp),it could be observed that the GUS staining was significantly increased,and the activity of GUS was also significantly increased.These results indicate that the promoter has transcriptional activity starting from P3(546 bp),and the promoter length of the 546 bp upstream of the initiation codon ATG is essential for driving the expression of the downstream GUS gene.We can also infer core elementof pGhRablld3 is located between the P2(385 bp)and P3(546 bp),may be a MYB transcription factor biriding site(492 bp)or a MADS box transcription factor binding site(417bp).In addition,we found that the upstream sequence of the 546 bp also contains regulatory elements that enhance the expression of downstream genes.The promoter of GhRab11d3 can be induced by three hormones(including GA,IAA,and 6-BA),but not by ABA.Finally,cotton ovulesare bombarded for gene gunthrough the five deletion promoter plasmid vector.When the fibersappear,they are stained with GUS.It was found that P3,P4 and P5 vectors had a certain GUS staining,which once again proved that GhRah11d3 played a certain function in the development of fiber.更多还原