棉花茉莉酸信号负调控因子GhJAZ2的克隆及功能分析

发布时间：2018-07-05 10:24

本文选题：棉花 + 纤维　；参考：《华中农业大学》2016年博士论文

【摘要】：棉花作为一种重要的经济作物,其纤维在纺织、民用和工业原料等方面都有重要的价值,是一种重要的经济作物。同时棉花纤维作为胚珠表皮细胞突起发育而成的单细胞纤维,是研究细胞分化的很好的材料。众所周知,纤维突起的数目和纤维的长度是构成棉花产量的重要因素。根据已有报道,影响棉花纤维的突起与发育的因素有很多,如:赤霉素、生长素、乙烯、茉莉酸等植物激素以及MYB类、HD-ZIP类以及其他类型的转录因子等。本实验室前期的研究表明高浓度的茉莉酸会抑制纤维的突起,但适量的浓度又能促进纤维的伸长。并且JAZ基因在纤维发育的早期优势表达,说明茉莉酸在纤维发育过程中具有重要的作用。基于这些结果我们进行了如下的研究:1.合适浓度的茉莉酸(JA)促进纤维的突起为了研究JA对纤维发育的影响,我们检测了-3 DPA(Days Post Anthesis)到8 DPA的纤维及胚珠的混合样中的JA的含量。结果显示-1 DPA样品中的JA含量最高,到0 DPA的时候有所降低,但都高于其他时期的样品中的JA含量。说明JA在纤维发育早期起关键作用。因此我们检测了不同突变体(GZnn、GZNn、n2、XinWX和Xu142 fl)和野生型(Xu142和TM-1)材料0 DPA胚珠的JA的含量,发现在突变体里的JA的含量低于野生型。进一步的胚珠培养实验发现在0.001μM的条件下,0 DPA的表皮上纤维细胞的突起数目比不添加JA的对照及其他浓度的处理有所增加。因此我们认为JA对纤维的发育存在剂量效应,合适浓度的JA能够促进纤维的突起。2.棉花基因GhJAZ2的克隆和序列分析根据NCBI的序列信息,我们克隆了JA信号传导路径上的关键基因JAZ(ES802380)。其编码的蛋白质包含JAZ家族高度保守的TIFY结构域和C-端的Jas结构域。将ES802380与拟南芥的JAZ家族的蛋白进行比对发现,其与AtJAZ1与AtJAZ2的蛋白最同源,其中与AtJAZ1相似度为33.21%,与AtJAZ2相似度为43.28%,因此将该基因命名为GhJAZ2。基因组织表达分析发现GhJAZ2在根、下胚轴、花器官和-1 DPA的胚珠里高量表达。3.在棉花中超表达GhJAZ2基因抑制纤维长绒和短绒的突起,以及长绒的伸长。为了验证GhJAZ2基因在棉花发育过程中发挥的功能,我们构建了CaMV35S超表达载体(CaMV 35S::GhJAZ2)和干涉载体(CaMV 35S::iGhJAZ2)并转化棉花。多年的重复试验结果发现,不同超表达株系的纤维长度均比对照组变短,种子变小。扫描电镜的结果及统计数据显示超表达株系长绒和短绒突起的数目比对照少。综上所述,在棉花中超表达GhJAZ2引起长绒短绒的突起都变少,纤维变短,种子变小,但是干涉GhJAZ2无明显表型。4.GhJAZ2通过与GhMYB25-like、GhGL1、GhMYC2、GhWD40和GhJI1等互作抑制纤维的突起为了进一步研究GhJAZ2影响纤维发育的作用机理,利用酵母双杂技术以GhJAZ2作为诱饵蛋白筛选-2 DPA~4 DPA的酵母双杂文库,获得了将近80个阳性克隆。最终我们关注了其中的5个互作蛋白,它们分别是R2R3-MYB类转录因子GhMYB25-like和GhGL1、bHLH类转录因子GhMYC2、WD40重复蛋白家族GhWD40和一个未知功能蛋白GhJI1。并且运用BIFC的方法证实了GhJAZ2与这5个转录因子确实存在互作。5.GhJAZ2通过与GhMYB25-like互作抑制突起目前,BIFC验证的这5个互作蛋白中只有GhMYB25-like被证实参与纤维的突起,其他几个均没有相关文献报道。报道表明,降低GhMYB25-like的表达量之后会导致纤维的突起减少,甚至光籽的表型。同时我们对棉花CaMV35S::GhMYB25-like超表达株系的表型鉴定结果表明,提高GhMYB25-like表达量会使0 DPA胚珠的纤维突起的数目增加。因此,我们将GhJAZ2的超表达株系与GhMYB25-like超表达株系进行杂交,发现杂交F1代(GhJAZ2×GhMYB25-like)的0 DPA胚珠表面纤维突起的数目与GhJAZ2的超表达株系的突起数目没有差异。同时在烟草的原生质体瞬时转化体系中,验证了GhMYB25-like存在转录激活功能,并且在GhJAZ2存在的时候GhMYB25-like的转录激活功能受到了抑制。因此,我们认为GhJAZ2通过与棉花纤维起始关键基因GhMYB25-like互作并抑制了其转录激活功能,从而抑制了纤维的突起。6.棉花JAZ家族中存在功能冗余在GhJAZ2的RNAi株系中,长绒短绒的数目以及长绒长度均与野生型没有差异。为了解释该表型,我们参考已经测序的陆地棉基因组数据,共获得棉花JAZ家族的30个成员。随后通过基因表达热图分析发现其中3个基因(Gh_D07G0152,Gh_D10G0531和Gh_D05G0379)的表达模式与GhJAZ2相似,并且FPKM值是大于GhJAZ2的。进一步的进化树分析发现另外2个基因(Gh_D08G2564和Gh_D05G0352)与GhJAZ2最同源。JAZ家族基因在陆地棉TM-1的不同组织中的表达模式分析发现其中Gh_D01G1406在-1 DPA中有一个表达高峰。将上述的6个基因的全长分别克隆出来用酵母双杂的方法验证与GhMYB25-like、GhGL1、GhMYC2、GhWD40和GhJI1之间是否存在互作,结果表明只有Gh_D01G1406能够与这5个蛋白分别互作,其他的几个均不与这5个蛋白存在互作。并且我们检测了该基因在GhJAZ2的转基因株系中的表达量,发现在干涉株系中该基因的表达量有所上调。据此,我们认为棉花干涉的株系没有出现表型可能是由于JAZ家族中存在与GhJAZ2功能冗余的其他成员。
[Abstract]:As an important economic crop, cotton is an important economic crop in textile, civil and industrial materials. It is an important economic crop. At the same time, cotton fiber is a single cell fiber developed from the protuberance of the ovule epidermal cells. It is a very good material to study the differentiation of cells. Fiber length is an important factor in the production of cotton. According to the reports, there are many factors affecting the protuberance and development of cotton fiber, such as gibberellin, auxin, ethylene, jasmonic acid and other plant hormones as well as MYB, HD-ZIP and other types of transcription factors. The protuberance of the fiber was inhibited, but a proper concentration could promote the elongation of the fiber. And the expression of JAZ gene at the early stage of fiber development showed that jasmonate played an important role in the development of fiber. Based on these results, we have studied the following results: 1. the appropriate concentration of jasmonic acid (JA) promotes the protruding of fiber in order to study the JA pair The effect of fiber development, we detected the content of JA in the mixed samples of -3 DPA (Days Post Anthesis) to 8 DPA fibers and ovules. The results showed that JA content in -1 DPA samples was the highest, and decreased at the time of 0 DPA, but higher than the JA content in the samples of other periods. The contents of JA of 0 DPA ovules of different mutants (GZnn, GZNn, N2, XinWX and Xu142 FL) and wild type (Xu142 and TM-1) materials were detected. The JA content in the mutant was found to be lower than that in the wild type. Further ovule culture experiment found that the number of fibrous cells on the surface of the surface of the 0 epidermis was compared with that of the 0.001 micron M. The treatment of his concentration has increased. Therefore, we think JA has a dose effect on the development of fiber. A suitable concentration of JA can promote the cloning and sequence analysis of the.2. cotton gene GhJAZ2 of the fiber. According to the sequence information of NCBI, we cloned the key gene JAZ (ES802380) on the JA signal transduction pathway. The encoded protein contains JA. The highly conserved TIFY domain of the Z family and the Jas domain of the C- end. Compared the protein of ES802380 to the Arabidopsis JAZ family, it was found that it was the most homologous to the proteins of AtJAZ1 and AtJAZ2, in which the similarity between AtJAZ1 and AtJAZ2 was 33.21% and the AtJAZ2 similarity was 43.28%. Therefore, the gene was named as the GhJAZ2. gene tissue expression analysis to find GhJAZ2 in The high expression of.3. in the ovule of the root, hypocotyl, flower organ and -1 DPA in the ovule of the cotton was overexpressed by the GhJAZ2 gene to inhibit the protuberance of fiber long velvet and short velvet, as well as the elongation of long velvet. In order to verify the function of the GhJAZ2 gene in the development of cotton, we constructed the CaMV35S overexpression vector (CaMV 35S:: GhJAZ2) and the interference carrier (CaMV 35S:: iGh). JAZ2) and transformation of cotton. Many years of repeated test results showed that the fiber length of different hyper expression lines became shorter and the seeds were smaller than those of the control group. The results of scanning electron microscopy and statistics showed that the number of overexpressed lines and short fleece projections was less than that of the control. In summary, the overexpression of GhJAZ2 in cotton flowers caused the changes of the short velvet. Less fiber, shorter fibers and smaller seeds, but the interference GhJAZ2 has no obvious phenotypic.4.GhJAZ2 through the interaction with GhMYB25-like, GhGL1, GhMYC2, GhWD40 and GhJI1 to inhibit the fiber development in order to further study the mechanism of GhJAZ2 affecting fiber development, using yeast double heterozygosity to select GhJAZ2 as bait protein for screening -2 DPA~4 DPA. Nearly 80 positive clones were obtained. Finally, we paid attention to 5 interactivity proteins, which are R2R3-MYB class transcription factors GhMYB25-like and GhGL1, bHLH transcription factor GhMYC2, WD40 repeat protein family GhWD40, and an unknown functional protein GhJI1. and confirmed that GhJAZ2 and these 5 transcription factors are true. The existence of interaction.5.GhJAZ2 by interacting with GhMYB25-like and inhibiting protuberance at the present time, only GhMYB25-like in the 5 interacted proteins verified by BIFC has been confirmed to be involved in the protuberance of the fiber, and the other few are not reported in the relevant literature. The results of phenotypic identification of cotton CaMV35S:: GhMYB25-like overexpressed strain showed that increasing GhMYB25-like expression could increase the number of fibrous protrusions in 0 DPA ovules. Therefore, we hybridize the overexpression strain of GhJAZ2 with the GhMYB25-like overexpression strain, and found the 0 DPA ovule surface fiber of the F1 generation (GhJAZ2 * GhMYB25-like). The number of vascular protrusions is not different from the number of GhJAZ2 overexpressed strains. Meanwhile, in the transient transformation system of protoplasts of tobacco, GhMYB25-like has a transcriptional activation function and the transcriptional activation function of GhMYB25-like is suppressed when GhJAZ2 exists. Therefore, we believe that GhJAZ2 is used to play with cotton fibers. The key gene GhMYB25-like interacts and inhibits its transcriptional activation function, which inhibits the fibrous protuberance of the.6. cotton JAZ family with functional redundancy in the RNAi line of the GhJAZ2. The number of long velvet and the length of the long velvet are not different from that of the wild type. In order to explain the phenotype, we refer to the genome number of the land cotton that has been sequenced. A total of 30 members of the cotton JAZ family were obtained. Subsequently, the expression pattern of 3 genes (Gh_D07G0152, Gh_D10G0531 and Gh_D05G0379) was found to be similar to GhJAZ2, and the FPKM value was greater than GhJAZ2. Further evolutionary tree analysis found that the other 2 genes (Gh_D08G2564 and Gh_D05G0352) were the most homologous to GhJAZ2. The expression pattern analysis of AZ family gene in different tissues of upland cotton TM-1 found that Gh_D01G1406 had an expression peak in -1 DPA. The whole length of the 6 genes was cloned separately to verify the interaction between GhMYB25-like, GhGL1, GhMYC2, GhWD40 and GhJI1 with yeast double heterozygosity. 406 can interact with these 5 proteins separately, and the others do not interact with the 5 proteins. And we detected the expression of the gene in the GhJAZ2 transgenic line and found that the expression of the gene was up in the interference strain. Accordingly, we think that the phenotype of the cotton stem related strains may not be due to the JAZ family. There are other members in the family with the redundancy of the GhJAZ2 function.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562

【参考文献】