植物双组分信号系统调控雌配子体发育的研究及其相关基因家族在白菜中的鉴定、进化和表达分析

发布时间：2018-06-08 10:23

  本文选题：雌配子体 + 胚囊　； 参考：《浙江大学》2016年博士论文

【摘要】：被子植物具有雌、雄两种配子体,其生活周期是二倍体孢子体和单倍体配子体世代交替的过程,其中,雌配子体(胚囊)在植物花粉管引导、受精和受精后的种子发育等生殖过程扮演着关键角色。雌配子体的发育是非常重要的发育事件之一,在基础理论研究和农业育种工作中具有重要意义,一直是植物生殖发育生物学研究的热点。研究表明,双组分信号系统(Two-component system, TCS)介导的磷酸化传递是植物调节细胞内信号转导的主要机制之一。被子植物具有包括组氨酸激酶(Histidine kinase, HK)、组氨酸磷酸转移蛋白(Histidine phosphotransferprotein,HP)和反应调节因子(Response regulator, RR)的多步磷酸化传递的双组分信号系统。目前对植物双组分信号系统功能的研究主要集中在逆境胁迫和营养生长方面,对生殖发育尤其是TCS对雌配子体发育和细胞命运分化的作用还知之甚少。为进一步揭示植物双组分信号系统与雌配子体发育和细胞命运决定的关系,本研究对模式植物拟南芥胚囊发育过程中的双组分信号进行了检测,并分析了参与双组分信号转导的组氨酸激酶基因在胚囊中的表达模式,重点研究了细胞分裂素受体对功能大孢子形成的调控以及CKI1 (cytokinin-indepenndent 1)基因与雌配子体细胞命运决定的关系。同时,为深入研究白菜中的双组分信号系统,基于白菜全基因组对白菜IPTs (isopentenyl transferases)和(CKXs (cytokinin oxidase/dehydrogenases)基因家族、TCS基因以及CRFs (cytokinin response factors)基因家族进行了鉴定,并对上述基因家族的基因组信息、蛋白特征、进化和表达模式等方面进行了研究,为今后开展其功能的分析奠定了基础。主要研究结果如下：(1)利用TCSnpro::NLS-3XeGFP marker对拟南芥胚囊发育过程中双组分信号的动态分布进行了观察,结果表明,双组分信号从胚囊发育的功能大孢子时期到成熟期在雌配子细胞中均存在,暗示双组分信号系统在雌配子体发育过程中具有重要的功能。对所有能够参与双组分信号转导的7个组氨酸激酶基因在胚囊发育过程中启动子活性的分析结果表明,除AHK2外,在胚囊中AHK1、 AHK3、AHK4、AHK5、CKI1和ETRl均检测到表达信号,且CKI1表达相对较高。对ahk2 ahk3 ahk4三突变体的观察结果表明,AHK2,AHK3和AHK4在调控功能大孢子发育时存在功能冗余。(2)拟南芥ckil-9/+突变体表现出雌配子体的败育表型。对胚珠DIC观察表明,突变体胚囊中两个极核往往不能正常融合,还伴随着未发生降解退化的三个反足细胞核从胚囊合点端向珠孔端的迁移。构建了雌配子体细胞特异表达的单1narker和双marker,并将上述marker通过拟南芥浸花转化法转入ckil-9,/+突变体中,结果表明,突变体胚囊中反足细胞和中央细胞的细胞命运彻底丢失,三个反足细胞和两个未融合的极核表现为卵细胞或者助细胞的细胞命运。对CKI1蛋白在胚囊发育过程中的定位分析表明,CKIl从胚囊发育的FG4时期开始表现出合点端的极性定位,这种极性定位可能是通过基因转录后的调节机制实现的。利用ES1启动子在胚囊中过表达CKIl基因,结果显示,CKI1在卵细胞和助细胞中的异位表达能够将卵细胞和助细胞的细胞命运转变成中央细胞的细胞命运。基于上述结果提出了雌配子体细胞命运决定的两种模型。(3)对,IHPI～AHP5在成熟胚囊中启动子活性的分析结果表明,AHP1基因在中央细胞和助细胞中表达,AHP3基因在中央细胞中特异表达,AHP2和AHP5基因在中央细胞、卵细胞、助细胞和反足细胞中均有表达,而AHP4基因在胚囊中不表达。随后构建了ahp多突变体并对多突变体的种子发育和胚囊发育进行了研究,结果显示,ahp2-2 ahp3 ahp5-2/+三突变体表现出与ckil-9/+相似的表型,AHP2、AHP3和AHP5共同作用于CKIl基因的下游调控雌配子体的发育。(4)对植物HK家族蛋白氨基酸序列的比对发现,CKI1在HK结构域和HATPase结构域中分别存在着氨基酸序列为SHD和GLGLG的特异motif,可以作为判定CKI1的重要依据。通过overlap extension PCR技术对CKI1结构域的替换研究了CKI1的结构域与CKI1基因功能的关系,结果表明,CKI1的HK结构域和HATPase结构域而非Rec结构域影响CKI1基因的功能。另外,CKI1蛋白亚细胞定位的研究结果显示,CKI1的N-端区域决定着CKI1蛋白在内质网上的定位。(5)对不同长度的CKI1启动子的研究表明,CKIl的核心启动子元件位于第一个内含子中。利用酵母单杂技术对CKI1上游转录调控因子进行了筛选和鉴定,最终筛选获得了7个在中央细胞中表达的转录因子基因。(6)在白菜基因组中分别鉴定到了13个IPTs基因和12个CKXs基因,并对其基因组信息和蛋白特征做了分析。多序列比对、保守结构域和系统进化分析结果表明,白菜IPTs和CKXs都可以分为Ⅰ、Ⅱ、Ⅲ和IV四个亚组。选取白菜、拟南芥和琴叶拟南芥三个十字花科植物中的IPTs和CKXs做了基因组层面的比较分析,发现其基因存在片段重复和串联重复现象,并通过计算直系同源基因和旁系同源基因的Ks和Ka值对基因的进化模式做了分析。结果显示,白菜中旁系同源基因的Ks值明显比白菜、拟南芥和琴叶拟南芥中直系同源基因的Ks值要小,同时,白菜在大约26～33MYA与拟南芥分离开。另外,通过qRT-PCR技术对白菜IPTs和CKXs在不同器官中表达模式的分析,筛选出了一些器官特异表达的基因。对白菜IPTs和CKXs在干旱胁迫和盐胁迫条件下以及对外源细胞分裂素和脱落酸响应模式开展研究,获得了一些参与非生物胁迫和激素调控网络的候选基因。(7)对白菜中的双组分信号元件进行了鉴定,共找到了85个TCS基因成员,包括20个HK基因、8个HPs和57个RRs,并对其基因组信息和蛋白特征做了分析。多序列比对、保守结构域和系统进化分析结果表明,HKs可以分为细胞分裂素受体亚家族、AHKl亚家族、AHK5亚家族、CKI1亚家族、乙烯受体亚家族和光敏色素亚家族。RRs可以分为Type-A RR、Type-B RR、Type-C RR和pseudo-RR四大类。我们分析和比较了白菜和拟南芥中TCS基因的片段重复和串联重复现象,并通过计算直系同源基因和旁系同源基因的Ks和Ka值对基因的进化模式做了分析。另外,通过qRT-PCR技术对白菜TCSs在不同器官中的表达模式做了分析,筛选获得了一些器官特异表达的基因。此外,还研究了白菜TCSs对干旱胁迫和盐胁迫的响应特征,以及对外源细胞分裂素、生长素和脱落酸的响应特征。(8)在白菜基因组中鉴定出了281个AP2/ERF超级基因家族成员,并对其保守结构域和进化树进行了分析,结果表明,白菜AP2/ERF超级家族可以分为AP2家族、RAV家族和ERF家族,其中ERF家族又可以进一步分为IREB亚家族和ERF亚家族,包括Ⅰ～Ⅺ 13个小组。对小组Ⅵ和VI-L中的21个CRFs做了重点研究。白菜CRFs可以分为Ⅰ～Ⅴ5个分支,编码A、B和C三种类型的蛋白。利用qRT-PCR技术对白菜CRFs在不同器官中表达模式开展研究,结果表明,CRFs在不同器官中是遍在表达的,但不同部位的表达量存在一定差异。通过研究CRFs对干旱胁迫和盐胁迫的响应模式以及对外源细胞分裂素和脱落酸的响应模式,筛选获得了一批参与非生物胁迫和激素调控网络的候选基因。研究结果能使我们从一个新的视角理解植物的雌配子体的发育过程,同时也为充分理解经济作物有性生殖的分子机制,完善芸薹属蔬菜作物的生殖生物学研究基础,有效调控作物育性,实现优质高产、高效繁殖提供新的理论依据和技术支持。
[Abstract]:The angiosperms have two gametophytes of female and male, whose life cycle is the process of alternation of diploid sporophyte and haploid gametophyte, in which the female gametophyte (embryo sac) plays a key role in the reproductive process, such as the pollen tube, fertilization and fertilization, and the development of the female gametophyte is one of the most important developmental events. The research shows that Two-component system (TCS) mediated phosphorylation is one of the main mechanisms for regulating intracellular signal transduction in plants. The angiosperms include histidine kinase, which includes histidine kinase. (Histidine kinase, HK), histidine phosphate transfer protein (Histidine phosphotransferprotein, HP) and reactive modulating factor (Response regulator, RR) multistep phosphorylation signaling system. The current research on the function of plant dual component signal system is focused on stress and nutritional growth, especially for reproductive development. The effect of TCS on the development of female gametophyte and cell fate differentiation is little known. In order to further reveal the relationship between the plant dual component signal system and the development of female gametophyte and cell fate, this study examined the two component signals in the development of the embryo sac of the model plant Arabidopsis thaliana, and analyzed the involvement of the two component signals. The expression pattern of the histidine kinase gene in the embryo sac focuses on the regulation of the cytokinin receptor on the formation of functional megaspore and the relationship between the CKI1 (cytokinin-indepenndent 1) gene and the fate determination of the female gametophyte. IPTs (isopentenyl transferases) and (CKXs (cytokinin oxidase/dehydrogenases) gene family, TCS gene and CRFs (cytokinin response factors) gene family were identified, and the genome information, protein characteristics, evolution and expression patterns of the above gene family were studied in order to carry out their functional analysis in the future. The main research results are as follows: (1) the dynamic distribution of two component signals during the development of the embryo sac of Arabidopsis thaliana was observed by TCSnpro:: NLS-3XeGFP marker. The results showed that the two component signals existed from the functional megaspore to the mature stage of the embryo sac, suggesting the dual component signal system in the embryo sac. The analysis of the promoter activity of all 7 histidine kinase genes involved in the double component signal transduction of the 7 histidine kinase genes in the embryo sac development showed that the expression signals were detected in the embryo sac, including AHK1, AHK3, AHK4, AHK5, CKI1 and ETRl in the embryo sac, and the expression of CKI1 was relatively high. The observation of AHK4 three mutant showed that AHK2, AHK3 and AHK4 had functional redundancy in regulating the development of the functional megaspore. (2) the ckil-9/+ mutant of Arabidopsis showed the abortive phenotype of the female gametophyte. The observation of the ovule DIC showed that the two polar nuclei in the mutant embryo sac often did not merge normally, and were accompanied by three anti degradation degradation. The cell nuclei migrated from the end of the embryo sac to the end of the pearl hole. The single 1narker and double marker expressed by the female gametophyte cells were constructed, and the marker was transferred into the ckil-9, / + mutant by the Arabidopsis thaliana transformation method. The results showed that the fate of the antipodal and Central cells in the mutant embryo sac was completely lost, and three antipodal cells were lost. And two non fused polar nuclei show the fate of the egg cells or the cells of the helper cells. Location analysis of the CKI1 protein in the development of the embryo sac indicates that CKIl begins to show the polar location of the junction end from the FG4 period of the embryo sac development, which may be realized through the regulation mechanism after the gene transcription. Using the ES1 promoter. The CKIl gene was overexpressed in the embryo sac, and the results showed that the ectopic expression of CKI1 in the egg and the helper cells could transform the fate of the egg and the helper cells into the cell fate of the central cell. Based on the above results, two models of the fate of the female gametocyte cells were proposed. (3) the promoter activity of IHPI ~ AHP5 in the mature embryo sac. The results showed that the AHP1 gene was expressed in the central and helper cells, and the AHP3 gene was expressed specifically in the central cells. The AHP2 and AHP5 genes were expressed in the central, oocyte, helper and antipodal cells, and the AHP4 gene was not expressed in the embryo sac. Then, the multiple AHP mutants were constructed and the seed development and embryo of the multiple mutants were developed. The results showed that the ahp2-2 ahp3 ahp5-2/+ three mutant showed a similar phenotype to ckil-9/+, and AHP2, AHP3 and AHP5 co regulated the development of the female gametophyte in the lower reaches of the CKIl gene. (4) the comparison of the amino acid sequence of the plant HK family protein found that CKI1 existed in the HK domain and the HATPase domain respectively. The base acid sequence is a specific motif of SHD and GLGLG, which can be used as an important basis for determining CKI1. The relationship between the domain of CKI1 and the function of CKI1 gene is studied by the replacement of CKI1 domain by overlap extension PCR technology. The results of the localization of white subcells show that the N- terminal region of CKI1 determines the localization of CKI1 protein on the endoplasmic reticulum. (5) the study of different lengths of CKI1 promoter indicates that the core promoter element of CKIl is located in the first intron. Using yeast single heterozygosity to screen and identify the upstream transcriptional regulators of the CKI1 and the final screening. 7 transcriptional factor genes expressed in central cells were selected. (6) 13 IPTs and 12 CKXs genes were identified in Chinese cabbage genome, and their genomic information and protein characteristics were analyzed. Multiple sequence alignment, conservative domain and phylogenetic analysis showed that both IPTs and CKXs can be divided into I, II, and III. A comparative analysis of IPTs and CKXs in cabbage, Arabidopsis and three cruciferous plants of Arabidopsis thaliana from Chinese cabbage, Arabidopsis, and Arabidopsis thaliana was made by comparison and analysis of genomic level. It was found that the gene existed in fragment repetition and series duplication, and the evolution patterns of the genes were analyzed by calculating the Ks and Ka values of the direct homologous and accessory homologous genes. The results showed that the Ks value of the homologous genes in Chinese cabbage was obviously smaller than that of Chinese cabbage, Arabidopsis and Arabidopsis thaliana in Arabidopsis thaliana. At the same time, Chinese cabbage was separated from Arabidopsis in about 26 to 33MYA. In addition, some organ specific tables were screened by qRT-PCR technology to analyze the pattern of IPTs and CKXs in different organs of Chinese cabbage. The genes of IPTs and CKXs were studied under drought stress and salt stress, and the response patterns of external cytokinin and abscisic acid were studied. Some candidate genes involved in the abiotic stress and hormone regulation network were obtained. (7) a total of 85 TCS gene members were identified by identification of two component signal components in Chinese cabbage. 20 HK genes, 8 HPs and 57 RRs were included, and their genomic information and protein characteristics were analyzed. Multiple sequence alignment, conservative domain and phylogenetic analysis showed that HKs could be divided into the cytokinin subfamily, the AHKl subfamily, the AHK5 subfamily, the CKI1 subfamily, the ethylene receptor subfamily and the photosensitive subfamily.RRs. It is divided into four categories: Type-A RR, Type-B RR, Type-C RR and pseudo-RR. We analyzed and compared the repeat and tandem repeats of TCS gene in Chinese cabbage and Arabidopsis, and analyzed the evolution patterns of the gene by calculating the Ks and Ka values of the direct homologous and accessory homologous genes. The expression patterns in the same organs were analyzed, and some organ specific genes were screened. In addition, the response characteristics of Chinese cabbage TCSs to drought stress and salt stress, as well as the response characteristics of external cytokinin, auxin and abscisic acid were also studied. (8) 281 AP2/ERF supergene families were identified in the cabbage genome. The analysis shows that the conserved domain and the evolutionary tree are analyzed. The results show that the AP2/ERF superfamily can be divided into AP2 family, RAV family and ERF family, and the ERF family can be further divided into IREB subfamily and ERF subfamily, including 13 groups I to ERF, and 21 CRFs in group VI and VI-L. It can be divided into 5 branches of I to 5, encoding three types of protein, A, B and C. Using qRT-PCR technology to study the expression patterns of CRFs in different organs. The results show that CRFs is expressed in different organs, but there are certain differences in the expression of different parts. Through the study of CRFs response to drought stress and salt stress As well as the response patterns of external cytokinin and abscisic acid, a number of candidate genes involved in the network of abiotic stress and hormone regulation are screened. The results can enable us to understand the development of the female gametophyte in plants from a new perspective, and to fully understand the molecular mechanism of sexual reproduction of the economic crops, and improve the molecular mechanism of the sexual reproduction of the economic crops. The research foundation of reproductive biology of Brassica vegetable crops provides a new theoretical basis and technical support for effectively regulating crop fertility and achieving high quality, high yield and high efficiency reproduction.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q944.4;Q943.2
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本文编号：1995473