【作者】 许俊强；

【导师】 王小佳； 宋明；

【作者基本信息】 西南大学 ， 蔬菜学， 2014， 博士

【摘要】 花粉萌发(Pollen Germination, PG)及花粉管伸长(Pollen Tube Growth, PTG)是开花植物有性生殖的重要阶段。Ca2+信号系统与激素在植物的花发育(成花诱导、花芽分化和开花调控)、有性生殖(研究主要集中在花粉萌发和花粉管生长)、逆境生理等方面都起着非常重要的作用。目前对Ca2+相关的信号传递机制的研究中发现有Ca2+/CaM[包括类CaM蛋白(CMLs)]、 Ca2+/CDPK和Ca2+/CBL三类钙信号系统。他们都含有不同个数的EF-hands结构域,EF-hands具有高亲和力以结合Ca2+。植物体中除了保守的CaMs家族,还有CMLs家族,在钙信号传导中也可能具重要作用。对拟南芥基因组的研究发现,其基因组含有7个CaMs和50个CMLs,其中5个CaMs和19个CMLs是在PG和PTG试验中发现的,这些基因中有4个基因(CML39、CML49、CML3和CML16)在PG过程中上调表达,CML49也在PTG中转录水平增加。植物物种中每类钙信号系统均为家族蛋白,大部分钙结合蛋白生理作用还未清楚,因此对钙信号系统家族成员功能的研究可能成为研究植物生长发育过程的热点内容。本试验以自交不亲和材料E1和F1为材料,通过双向电泳筛选得到钙感受蛋白CML49,通过同源克隆及RACE技术从花粉中克隆了CML49基因,从柱头中克隆得到SRK7基因。构建了CML49及其及突变体、SRK7及其截短体的原核表达载体和酵母双杂交重组表达载体,并通过β-半乳糖苷酶活性检测相互作用强度,筛选与鉴定CML49与SRK7相互作用及其蛋白互作的结构域。从番茄中扩增得到花粉特异性启动子LAT52,构建了LAT52启动下的CML49反义表达载体,对甘蓝进行转化,从而验证CML49基因的生物学功能。本研究希望能为CML49基因在花粉萌发和花粉管伸长的研究,及研究钙调蛋白CML49在自交不亲和过程的作用提供参考。主要试验结果如下：1.双向电泳筛选、确定钙感应蛋白CML49以结球甘蓝E1、F1为材料,提取花粉萌发前和萌发后的混合花粉总蛋白,总蛋白双向电泳后通过MALDI-TOF-MS鉴定分析差异点,质谱分析得到花粉萌发中表达下调的蛋白点CML49。2.结球甘蓝CML49基因的克隆及分析通过同源扩增及RACE扩增得到了CML49基因的全长序列1343bp,开放阅读框954bp,编码317个氨基酸残基,预测分子量大小为33.51kD,p1为6.93。经Smart-embl预测其含2个重要的EF-hand结构域；与拟南芥AtCML49和AtCML50的亲缘关系较近；CML49基因的不同器官的表达分析表明该基因在不同器官均有表达,且在茎中的表达量最高,在花粉中也有较高的表达量；CML49基因的qPCR分析表明其表达量在花粉萌发前约为萌发后的2.73倍,表明CML49基因在花粉萌发后表达下调。3.甘蓝CML49与SRK7相互作用检测以F1材料柱头总RNA逆转录合成第一链cDNA为模板,扩增得到SRK基因2118bp,经NCBI比对所得到的SRK序列为S7单倍型。利用同源重组技术,分别构建了CML49与SRK7的原核表达载体pCold I-CML49和pGEX-SRK7。融合蛋白体外表达纯化,P μLl-down表明两融合蛋白在体外能够进行相互作用。同时分别构建CML49与SRK7(不含信号肽)的酵母表达载体pGBKT7-CML49和pGADT7-SRK7。利用醋酸锂转化法将重组酵母质粒转化到感受态酵母中,得到酵母转化子Y2HGold (pGBKT7-CML49)和Y187(pGADT7-SRK7),经过毒性及自激活检测,发现无自激活和毒性现象。融合的二倍体酵母Y2HGold (pGBKT7-CML49)和Y187(pGADT7-SRK7)能在选择型培养基SD/-Leu/-Trp/AbA (DDO/A)、SD/-Ade/-His/-Leu/-Trp (QDO)上正常生长、且在SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA (QDO/X/A)变蓝色。β-半乳糖苷酶活性测定表明CML49与SRK7相互作用的酶活性18.44。综上说明,结球甘蓝CML49与SRK7蛋白能够相互作用。4.CML49与胞内激酶域(iSRK7)、胞外域(eSRK7)相互作用检测从全长SRK7上亚克隆了胞内激酶域(不含跨膜域,iSRK7)750bp和胞外域(不含信号肽,eSRK7)1245bp2个SRK7亚结构域。构建原核表达载体pGEX-iSRK7和pGEX-eSRK7,进行原核表达,纯化融合蛋白,并进行体外相互作用检测,结果表明CML49能与eSRK7发生相互作用且相互作用强度很强,而与iSRK7没有检测到发生相互作用。为进一步验证上述结果,分别构建酵母重组表达质粒pGBKT7-CML49、pGADT7-iSRK、pGADT7-eSRK7及互换载体pGADT7-CML49、pGBKT7-iSRK7、pGBKT7-eSRK7,并转化对应的酵母菌株。结果显示二倍体酵母Y2HGold (pGBKT7-CML49)×Y187(pGADT7-eSRK7)和Y187(pGADT7-CML49)×Y2HGold (PGBKT7-eSRK7),在QDO/X/A培养基上长出蓝色菌落且生长速度很快,反应较为强烈,同时激活了酵母的报告基因AURl-C、HJS3、ADE2、MEL1。而Y2HGold (pGBKT7-CML49)×Y187(pGADT7-iSRK7)和Y187(pGADT7-CML49)×Y2HGold (pGBKT7-iSRK7),生长速度缓慢,几乎观察不到蓝色菌斑产生。通过β—半乳糖苷酶活性测定表明：CML49与eSRK7能够相互作用且强度较强,而CML49与iSRK7相互作用较弱。表明CML49与SRK7的相互作用的核心区域为胞外域,而不是胞内激酶域iSRK7。5. CML49EF-hand突变体与SRK7、eSRK7及iSRK7相互作用检测将CML49蛋白两个EF-hand结构域中重要的氨基酸突变,使EF1的谷氦酸Glu-170(E)突变为谷氨酰胺Gln-170(Q),然后突变CML49使EF2的谷氨酸Glu-236(E)突变为谷氨酰胺Gln-236(Q),最后突变两者,构建3种突变体,分别命名为CML49-1-、CML49-2-和CML49-12-三种突变体。构建原核表达载体pCold I-CML49-1-、pCold I-CML49-2-、pCold I-CML49-12-及pGEX-iSRK7、pGEX-eSRK7。进行原核表达,纯化融合蛋白,并进行体外相互作用检测,表明CML49突变体均不能与SRK7、iSRK7和eSRK7发生相互作用。同时构建酵母表达载体pGBKT7-CML49-1-、pGBKT7-CML49-2-、pGBKT7-CML49-12-及pGADT7-SRK7、pGADT7-iSRK7、pGADT7-eSRK7。结果进一步支持了原核表达体外相互作用检测结果。6.番茄花粉特异性启动子LAT52的克隆及功能分析根据番茄(S. lycopersicum)花粉特异性启动子LAT52序列(GenBank:X15855)设计引物,从番茄基因组DNA中扩增得到花粉特异性启动子核心序列608bp。序列分析表明其中含有多个花粉特异性元件。将LAT52启动子替换35S启动子连接到双元表达载体pBI121,命名为pBI-LAT52,转化农杆菌GV3101感受态细胞。蘸花法侵染拟南芥花序,获得T0代种子,抗性筛选后获得T1代拟南芥转基因植株。同时侵染甘蓝幼苗下胚轴。以pBI121载体作为阳性对照,在适当的MS培养基上经预培养、侵染、共培养、脱菌、筛选培养基、分化培养基、成苗、移栽等,获得转化甘蓝植株。对甘蓝植株花蕾、花药及花粉进行GUS染色,与阳性对照和阴性对照相比,仅在花药和花粉中检测到蓝色,说明花粉特异性启动子LAT52仅能够在花粉中特异性表达,且该启动子可以在甘蓝花粉中特异性表达。7.甘蓝CML49反义基因表达载体构建对甘蓝反义转化根据CML49基因全长序列,克隆得到反义CML49基因,并结合植物双元载体pCAMBIA1302的多克隆位点,设计引物并添加适当酶切位点。构建反义表达载P1302-LAT-aCML49、反义p1302-35S-aCML49,空载体作为阳性对照,转化甘蓝幼苗下胚轴,在适当的MS培养基上经预培养、侵染、共培养、脱菌、筛选培养基、分化培养基、成苗、移栽等,获得转基因甘蓝反义植株。8.甘蓝CML49反义基因转化植株的分子检测及形态学观察提取转基因甘蓝植株基因组DNA,设计引物分别检钡T-DNA区域内的CaMV35S启动子、Hyg(Ⅱ)潮霉素抗性基因、LAT52启动子及反义目的基因片段。经PCR检测,均检测到正确的目的片段,初步说明T-DNA区域片段成功转化甘蓝。设计探针引物,经Southern blot分析进一步确定对甘蓝的转化,且以双拷贝整合到甘蓝植株基因组中。转基因植株甘蓝花粉原位萌发显示p1302-35S-aCML49和p1302-LAT-aCML49转基因植株柱头上萌发的花粉数量较少,且花粉管大部分不能正常生长或生长速度较慢,仅可以观察到少量的花粉管穿过柱头；而对照株花粉大量萌发,且花粉管数量多且花粉管能正常伸长向胚囊。这表明反义CML49能使花粉萌发率降低,花粉管伸长不能正常进行。花粉体外萌发试验显示反义p1302-35S-aCML49和p1302-LAT-aCML49转基因植株的花粉萌发率明显低于对照,且花粉管的长度也明显缩短。花粉萌发前后的定量表达分析表明转基因植株中该基因的表达量明显降低,说明CML49基因在花粉萌发和花粉管伸长过程中起重要作用。更多还原

【Abstract】 Pollen germination, along with pollen tube growth, are essential processes for the reproduction of flowering plants. Ca2+signal system and hormone play very important roles in plant flower development (include floral induction、 flower bud differentiation and flowering regulation), generative propagation (researches mainly focus on pollen germination and pollen tube growth) and adversity physiology. The three largest categories of proteins of Ca2+signaling systems in plants are the CaMs (calmodulins) and CMLs (CaM-like proteins), the CDPKs (Ca2+-dependent protein kinases), and the CBLs (calcineurin B-like proteins). They all contain the different amount of EF-hands structure to combine Ca2+with high affinity. In addition to conserved CaMs, plants possess an extended family of CMLs.They may also have important roles in calcium signaling. The Arabidopsis genome contains7CaM genes and50additional CML genes.Among these genes, four CML genes (CML39, CML49,CML3, and CML16) were up-regulated during PG,12genes (CaM7, CML49, CML3, CML16, et al.) increased their transcription during PTG. In spite of the potential importance in mediating plant calcium signaling, the physiological functions of the CaMs and CMLs remain largely unknown. So, the studies of the functions of family members in calcium signal systems may become the hot spots in the process of plant growth and development.In this study,we took Self-incompatible (SI) El and Fl as material, obtained calcium sensor protein CML49by two-dimensional electrophoresis, and gained CML49gene from pollen by homologous cloning and RACE, then obtained SRK7gene from stigma. We constructed prokaryotic expression vectors and yeast two hybrid recombinant expression vectors of CML49and its mutants、 SRK7and its truncations, and then detected their interaction strengthes by P-Galactosidase Assay.We cloned pollen-specific promoter LAT52from tomoto, and constructed antisense CML49expression voctors under LAT52promoter, transformed into cabbage. Finally, we verified the biology function CML49gene. We hope to provide references for CML49gene research in pollen germination and pollen tube elongation, and the roles of CML49in Self-incompatible process.The main res μLts were showed as follows:1. The2-D electrophoresis to screen and determine calcium protein CML49geneExtracted total protein before and after the pollen germination, Calmodnlin-like49(CML49) protein of cabbage (Brassica oleracea L. var. capitata) was identified in the process of pollen germination in cabbage line Fl by two-dimensional electrophosis of the pollen total protein.2. Cloning and analysis of CML49gene in cabbageWe got f μLl length of CML49gene sequence with1343bp by homologous amplification and RACE amplification, open reading frame (ORF) was954bp, encoded317amino acid residues, predicted the molec μLar weight33.51kD, pl was6.93.It contains two EF-hand structure, and has close genetic relationship with AtCML49and AtCML50; Relative expression analysis showed that CML49gene were expressed in various organs of cabbage, and has the highest amount of expression in stem, also has higher expression level in pollen; qPCR analysis of CML49showed that the expression level of before pollen germination was about2.73times than after germination, and show that it is down-reg μLated after the pollen germination.3. Interaction between CML49and SRK7in cabbageTake first chain cDNA of stigma total RNA as template from Fl material, we got SRK gene of2118bp, the SRK sequence we got was S7haploid type by BLAST in NCBI database. And then we constructed CML49and SRK7prokaryotic expression vector pColdI-CML49and pGEX-SRK7using homologous recombination technology, respectively. After expression and purification in vitro, the two fusion proteins could do interaction in vitro. Meanwhile, constructed pGBKT7-CML49and pGADT7-SRK7(excluding signal peptide) yeast expression vector, respectively. Transformed recombinant yeast plasmid into competent cell by lithium acetate method, we got Y2HGold (pGBKT7-CML49)and Y187(pGADT7-SRK7), it has no activation and toxicity after toxicity and the activation detection. Diploid yeast of Y2HGold (pGBKT7-CML49) and Y187(pGADT7-SRK7) can grow on SD/-Leu/-Trp/AbA (DDO/A), SD/-Ade/-His-Leu/-Trp (QDO),and change blue on SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA (QDO/X/A). β-galactose glucoside assay showed that enzymatic activity of interaction between CML49and SRK7was18.44. In a word, CML49protein can interact with SRK7protein in cabbage.4. Interactions between CML49and intracell μLar kinase domain (iSRK7), extracell μLar domain (eSRK7)We subcloned two SRK7domain structures [iSRK、eSRK7(excluding signal peptide)] from SRK7with750bp and1245bp, respectively. Constructed prokaryotic expression vector pGEX-iSRK7and pGEX-eSRK7, prokaryotic expression and purification of fusion protein, and tested their interactions in vitro, the res μLts showed that the CML49can interact with eSRK7, but we did not detect the interaction with iSRK7. In order to verify the res μLts above furtherly, we constructed yeast recombinant expression plasmids pGBKT7-CML49, pGADT7-iSRK7, pGADT7-eSRK7and exchange vectors pGADT7-CML49, pGBKT7-iSRK7, pGBKT7-eSRK7, respectively, and transformed into corresponding yeast strains. The res μLts show that the diploid yeast Y2HGold (pGBKT7-CML49)×Y187(pGADT7-eSRK7) and Y187(pGADT7-CML49)×Y2HGold (pGBKT7-eSRK7) grew fast on the QDO/X/A medium and change blue, reaction is relatively strong, and activate AURl-c, HIS3, ADE2, MELI the yeast’s report genes at the same time. while Y2HGold (pGBKT7-CML49)×Y187(pGADT7-iSRK7) and Y187(pGADT7-CML49)×Y2HGold (pGBKT7-iSRK7) the grew slow, and almost hardly see blue plaque.β-galactose glucoside assay showed that CML49can strongly interact with eSRK7, but CML49weakly interact with iSRK7. These above show that the interaction core region of SRK7with CML49was the extracell μLar domain(eSRK7), rather than intracellular kinase domain (iSRK7).5. Interactions between CML49EF-hand mutants and SRK7, eSRK7、iSRK7We made mutations of important amino acid in two EF-hand structure domain of CML49protein, changed glutamate Glu-170(E) to glutamine Gln-170(Q) in EF1, and then changed glutamate Glu-236(E) to glutamine Gln-236(Q) in EF2, and finally changed both, named CML49-1-, CML49-2-and CML49-12-, respectively. We constructed prokaryotic expression vector pCold I-CML49-1-, pCold I-CML49-2-, pCold I-CML49-12-and pGEX-iSRK7, pGEX-eSRK7. After expression and purification, and detected their interactions in vitro. The res μLts showed that CML49mutants can not do interactions with SRK7, iSRK7and eSRK7. Meanwhile,we constructed yeast expression vectors pGBKT7-CML49-1-、pGBKT7-CML49-2-、pGBKT7-CML49-12-and pGADT7-SRK7、pGADT7-iSRK7、pGADT7-eSRK7. The res μLts supported the interaction between prokaryotic expression in vitro furtherly.6. Cloning and functional analysis of pollen cell-specific promoter LAT52from tomatoAccording to the tomato (S. lycopersicum) pollen specific promoter LAT52sequences (GenBank:X15855), we cloned core pollen specific promoter sequences with608bp from tomato genome DNA. Sequence analysis showed that it contains many pollen specific components. Connect LAT52promoter to binary expression vector pBI121replaced35S promoter, named pBI-LAT52, and then transformed into Agrobacterium GV3101competent cell, infection Arabidopsis inflorescence, gained TO generation seeds, resistance screening after T1generation of Arabidopsis transgenic plants.Meanwhile infected cabbage seedlings, gained cabbage plants. GUS staining, compared with the positive control and negative control, we only detected blue in anther and pollen of cabbage, it showed that LAT52only can specific expression in pollen, and the LAT52promoter can specific expression in cabbage pollen.7. Expression vector construction of antisense CML49gene and transformation into cabbageAccording to CML49gene sequence, cloned sense and antisense CML49gens, and combined with the m μLtiple cloning site of plant binary vector pCAMBIA1302, designed primers with proper digested sites. We constructed p1302-35S-aCML49and p1302-LAT-CML49, and took pCAMBIA1302null vector as positive control. Infected cabbage seedling hypocotyl, on the proper MS culture medium, infection, trained, take off bacterium, filter medium and differentiation medium, into seedlings and transplanting, obtain transgenic cabbage plants.8. Molec μLar detection and morphological observation of antisense CML49gene in transgene cabbageExtracted genome DNA of transgenic cabbage, design primers of CaMV35S promoter,Hyg hygromycin resistance gene, LAT52promoter and antisense gene fragment in T-DNA area.We detected correct fragments of all after PCR, all above showed that T-DNA fragment transformed cabbage successf μLly. Design probes, the Southern blot analysis to further determine the conversion of cabbage, and integrated into the cabbage plant genome with double copies.Pollen germination of transgenic cabbage in situ showed that p1302-35S-aCML49and p1302-LAT-aCML49transgenic plants has less quantity of pollen germination on stigma, and the speed of most pollen tube can’t normal growth or grow slowly, only a small amount of pollen tube can be observed through the stigma; while compared to postive, pollen tube number and pollen tube elongation properly to blastophore.It showed that antisense CML49can reduce pollen germination rate, and pollen tube elongation unable to proceed normally. Pollen germination experiments in vitro showed that pollen germination rates of antisense pl302-35S-aCML49and p1302-LAT-aCML49transgenic plants are significantly lower than control, and the length of the pollen tube also reduced significantly. The expression analysises of pollen germination showed that the gene expressions in transgenic plants were lower significantly, suggest CML49gene plays important roles in the process of pollen germination and pollen tube growth.更多还原