【作者】 蒋明；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2007， 博士

【摘要】 在植物花粉发育过程中,大量基因参与小孢子发育、花粉壁构建、有丝分裂、减数分裂、花粉填充物合成等活动,这些基因高度有序的表达是获得可育花粉的必要条件和重要保证,基因突变或者环境条件改变都有可能影响花粉发育,甚至引起雄性不育。为研究白菜(Brassica campestris L.ssp.chinenesis Makino,syn.B.rapa ssp.chinenesis)Polima核质互作雄性不育系’Bcpol97-05A’、Ogura细胞质雄性不育系‘Bcogu97-06A’与其共同的保持系’Bcajh97-01B’蕾期基因表达差异,我们采用cDNA-AFLP技术和ATHl GeneChip研究三个材料蕾期基因的表达情况,分析并揭示在相同核背景下两胞质不育材料蕾期基因表达的异同,为花粉发育和植物雄性不育提供分子依据。在此基础上,我们利用RACEs(rapid amplification of cDNA ends)利同源克隆等方法从‘矮脚黄’白菜及近缘十字花科植物中克隆了一些重要基因。(1)利用cDNA-AFLP技术分析’Bcpo197-05A’、‘Bcogu97-06A’与’Bcajh97-01B’蕾期基因表达差异,获得了98条511性差异转录片段(transcript-derived fragments,TDFs),在‘Bcpol97-05A’与‘Bcajh97-OIB’花蕾特异表达61条,‘Bcpol97-05A’花蕾中特异沉默30条,‘Bcp0197-05A’花蕾特异表达的12条。‘Bcogu97-06A’与’Bcajh97-01B’花蕾存在差异TDFs有75条,其中在’Bcogu97-06A’花蕾中特异沉默的40条,‘Bcogu97-06A’花蕾特异表达的8条。98条阳性TDFs中,21条在保持系’Bcajh97-01B’中特异表达,4条仅在’Bcogu97-06A’花蕾中特异表达,8条仅在’Bcpol97-05A’花蕾中表达。这些TDFs代表的基因涉及细胞壁合成与调控、防卫与胁迫反应、电子传递和能量途径、普通新陈代谢、蛋白质代谢、信号转导、转录调控和转运通道等方面。(2)利用拟南芥Affymetrix全基因组芯片ATHl GeneChip研究’Bcpol97-05A’、‘Bcogu97-06A’和’Bcajh97-01B’蕾期基因表达差异,共得到688个差异表达基因,338个基因在两不育系中上调,352个基因在两不育系中下调。在上调基因中,63个基因为两不育系所共有,其中有大量的叶绿体、线粒体基因参与上调表达,‘Bcpol97-05A’材料特有的上调基因有94个,其中24个为叶绿体或线粒体基因,‘Bcogu97-06A’材料中特异上调基冈有116个,其中4个为叶绿体和线粒体基因:在338个下调基冈中,75个基因在两不育系中共同下调,在’Bcpol97-05A’中下调的基因有93个,‘Bcogu97-06A’材料中下调的基因有109个。借助拟南芥Affymetrix全基因组基因芯片,获得了比cDNA-AFLP技术更为丰富的差异表达信息。(3)以‘Bpol97-05A’和‘Bajh97-01B’为材料,根据cDNA-AFLP技术获得一条长约330 bp的特异序列P1708,RT-PCR验证确认该序列仅在不育白菜材料中表达,经测序和BLAST比对,发现该片段与叶绿体trnL和ndhJ基冈之间的一段序列完全一致,并有54 bp的重复序列。根据trnL和ndhJ基因两端的保守序列设计全和长引物,分别以不育白菜及其原保持系的cDNA为模板,获得了长约2,000 bp的序列,该序列包含trnL、trnF和ndhJ等三个基因的完整序列,比对两序列发现:不育白菜‘Bpol97-05A’中除108 bp的插入序列外,另有多个位点发生变异。(4)富含脯氨酸蛋白(proline-rich protein,PRP)是构成细胞壁的一类重要结构蛋白,在花粉发育过程中,也同样需要这类蛋白。根据cDNA-AFLP获得的差异表达片段B0610,利用RACEs技术住‘Bajh97-05B’中克隆到了基因全长,定名为白菜花药特异富含脯氨酸蛋白(Brassica campestmsAnther-specific Proline-rieh Protein,BcAPG)基因。基因编码区长1,731 bp,编码576个氩基酸,推导的蛋白质序列中,脯氨酸残基含量极高,占总氨基酸数量的22.4%。另外,我们从6种十字花科植物中克隆到了它的同源基因,所推导的氨基酸与NCBI中己知的PRP相比,序列存在较大差异,推测这些基因产物属于一类新的PRP。RT-PCR结果表明,该基因仅在可育的Ⅲ级、Ⅳ级和Ⅴ级花蕾中表达,其它各级花蕾及营养器官中均朱检测到,而且,在两个不育系中的所有花蕾均未见表达。进一步实验表明,仅在花约中检测到BcAPG的表达,而花丝、花瓣、花梓和花萼中均未见表达,推测可能在白菜花粉壁的构建中起着重要作用。(5)拟南芥MS2与雄性不育密切相关,基因芯片数据分析表明,三个白菜材料在MS2探针上存在差异,保持系’Bajh97-01B’中检测到较强的杂交信号,而两个不育系中信号缺失。利用同源克隆技术,分别从‘Bajh97-01B’叶片基因组DNA和混合花蕾cDNA中克隆到了MS2同源基因的DNA和cDNA全长,命名为白菜雄性不育2(Brassica campestris Male Sterility 2,BcMS2)基因。BcMS2的DNA全长为2,576bp,具8个外显子和7个内含子,编码区长1,851 bp,编码616个氨基酸,蛋白质C端含有一个微体目标信号序列Gla-Arg-Ala,该蛋白位点与微体特异结合。RT-PCR结果显示,BcMS2仅在可育材料的Ⅲ级花蕾中表达,其它各级花蕾及营养器官中均未能检测到,在两不育材料的所有部位都没有表达,进一步研究表明,BcMS2的表达具有花药特异性。(6)肌动蛋白是细胞骨架微丝系统的重要组成部分,在花粉发育及花粉管伸长过稗中,肌动蛋白的活动十分活跃,是花粉可育和花粉管顺利生长的重要保证。根据已知序列,设计全长引物,从混合花蕾cDNA和叶片基因组DNA中克隆到了各自的全长,基冈命名为BcACT(Brassica campestrisActin)基因。其DNA全长为1,704 bp,具3个内含子,编码区长度为1,134 bp,编码377个氨基酸。基因表达结果显示,BcACT基冈在保持系‘Bajh97-05B’和Polima不育系‘Bpol97-05A’的花蕾中表达,但表达模式存在差异,而在Ogura不育系‘Bogu97-06A’未见表达。此外,BcACT基冈的表达具有花药特异性,随着花约的成熟,表达量逐渐增加。(7)根据基因数据库已有的拟南芥profilin4及其同源基因设计简并引物,住白菜花蕾cDNA中成功克隆到前纤维蛋白基因,命名为白菜前纤维蛋白(Brassica campestrisProfilin,BcPRO)基因,基因库中的登陆号为EF492988。BcPRO编码区由405个核苷酸组成,编码134个氨基酸,推导的氨基酸序列与甘蓝型油菜和拟南芥的花粉前纤维蛋白分别有95%和92%的同源性。RT-PCR表明,BcPRO仅在保持系的Ⅲ级、Ⅳ级和Ⅴ级花蕾及Polima雄性不育系的Ⅲ级和Ⅳ级花蕾中表达,并且表达量逐级增大,而在莲座叶、花茎叶、茎、根及Ogura雄性不育系的所有部位均未能检测到。进一步研究发现,BcPRO在花药中表达,在萼片、花丝、雌蕊、花瓣等部位均未见表达。这些实验结果表明,BcPRO为花药特异基因,可能在发粉发育过程中起着重要的作用,并且在相同核背景下,有着不同的核-质互作模式。(8)基因芯片研究白菜材料蕾期基冈表达差异时,发现拟南芥的一个查尔酮合成酶基因探针的杂交信号有显著差异,保持系‘Bcajh97-01B’和Ogura细胞质雄性不育系‘Bcogu97-06A’有较强的杂交信号,而Polima核质互作不育系中没有检测到相应信号。根据基因数据库中已发布的拟南芥及其同源基因序列,设计了全长扩增引物,成功克隆出该基因,命名为白菜查尔酮合成酶(Brassica campestrisChalcone Synthase,BcCHS)基因。该基因在NCBI基因数据库中的登陆号为EF101136。BcCHS基因的编码区全长为1,188 bp,编码395个氨基酸,基因组DNA全长为1,263 bp,具一个长75 bp的内含子。BcCHS在保持系‘Bcajh97-01B’和Ogura细胞质雄性不育系‘Bcogu97-06A’的Ⅳ级、Ⅴ级花蕾及开放的花朵中表达,进一步研究表明,BcCHS在花药和花瓣中表达,在花朵的其它部位没能检测到。此外,在白菜白花突变体基因组DNA中克隆到BcCHS-wf,该基因与野生型白菜BcCHS基因相比,发现了两个突变位点,在推导的氨基酸序列中,相应位置编码精氨酸的AGA和编码丝氨酸的AGC均替换成了甘氨酸。更多还原

【Abstract】 During the process of pollen development,thousands of genes take part in activities as microspore development,pollen wall construction,mitosis,meiosis and synthesis of pollen materials.Spatiotemporal expression of these genes is essential for the formation of fertile pollen grains.Changes of environmental factors or gene mutation may affect pollen development or even cause male sterility.We adopted cDNA amplified fragment length polymorphism(cDNA-AFLP)technique and Affymetrix GeneChip to learn about genes expressed during flower bud development among three Chinese cabbage-pak-choi(Brassica campestris L.ssp.chinenesis Makino,syn.B.rapa ssp.chinenesis)materials,Polima cytoplasmic male sterile(CMS)line ’Bcpol97-05A’,Ogura CMS line ’Bcogu97-06A’and their co-maintainer line ’Bcajh97-01B’.On the basis of cDNA-AFLP and GeneChip results,we isolated some important genes from ’Aijiaohuang’ Chinese cabbage-pak-choi and its relative species by means of rapid-amplification of cDNA ends(RACEs)and homology-based cloning method.(1)By means of cDNA-AFLP,98 transcript-derived fragments(TDFs)were obtained.Their homologous genes participated in cell wall metabolism,defense response,electron transport or energy pathway,metabolism,protein metabolism,signal transduction,transcription and transport.61 TDFs expressed especially in flower buds of ’Bcpol97-05A’ and "Bcajh97-01B’,and 30 TDFs silenced in "Bcpol97-05A’.12 TDFs were detected in ’Bcpol97-05A’ and no expression in other two materials.There were 75 TDFs difference between ’Bcogu97-06A’ and "Bcajh97-01B’ and 40 were found silent in ’Bcogu97-06A’.8 TDFs expressed only in flower buds of "Bcogu97-06A’.(2)Total 688 differential expressed genes were obtained from three Chinese cabbage-pak-choi materials, ’Bcpol97-05A’,’Bcogu97-06A’ and ’Bcajh97-01B’,by Arabidopsis whole genomie ATH1 genechip.388 of them were down-regulated and 352 up-regulated in two male sterile lines.In those up-regulated genes,the two CMS line shared 63 genes and there are amount of mitochondrial and chloroplast genes.94 genes especially expressed in flower buds of "Bcpol97-05A’ and 24 of them were chloroplast and mitochondrial genes.Among 338 down-regulated genes,the two CMS materials shared 75 genes.There were 93 genes specially down-regulated in "Bcpol97-05A" flower buds and 109 genes in "Bcogu97-06A’.(3)By means of cDNA-AFLP,a fragment P1708 was amplified from cytoplasmic male sterile Chinese cabbage ’Bpol97-05A’ in Chinese cabbage-pak-choi,RT-PCR showed that this fragment was specifically expressed in male sterile material.Sequencing and Blast indicated that P1708 had 100%homolog with chloroplast gene between tree and ndhJ genes except a 54-bp repeat sequence.We designed the full length primers according to trnL and ndhJ gene region and a 2000-bp fragment was amplified using cDNA as templates.The sequencing results showed that the gene region trnL-ndhJ of cytoplasmic male sterile Chinese cabbage-pak-choi had a 54-bp repeat sequence and some sites variation.(4)A novel anther-specific proline rich protein gene,designated BcAPG(Brassica campestris anther-specific proline-rich protein gene),was isolated from male fertile Chinese cabbage-pak-choi ’Bajh97-05B’ by means of cDNA-AFLP and RACEs.BcAPG gene contained an open reading frame of 1,731 bp encoding 576 amino acids with high level of proline residue(22.4%).RT-PCR indicated that BcAPG expressed specifically in anthers of ’Bajh97-O5B’ but no detection in all tissues of two CMS lines, Polima CMS line ’Bpol97-05A’ and Ogura CMS line ’Bogu97-06A’.Homologous genes of BcAPG were isolated from other six colewort crops and sequence comparison was performed.(5)A male sterility gene homolog,designated BcMS2(Brassica campestris Male Sterility 2 gene),was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533.Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from B. napus revealed some differences in gene structure and evolution.The full genornic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns,more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stageⅢflower buds of male fertile Chinese cabbage-pak-choi ’Bajh97-01B’ and there were no detection in all organs of Polima CMS line ’Bpol97-05A’ and Ogura CMS line ’Bogu97-06A’.Furthermore,RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals,petals,filaments and pistils.These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.(6)Actin is an important material for construction of cell cytoskeleton microfibre systems.During pollen development and pollen tube elongation,the higher activity of actin protein is essential for both biological processes.We designed full length primer pairs according to known sequences and successfully isolated the gene which was assigned BcACT(Brassica campestris actin protein gene)from both mixed flower buds cDNA and genomic DNA of Chinese cabbage-pak-choi.BcACT is 1,704 bp in length with 3 introns.The complete cds of BcACT is 1,134 bp in length encoding 377 amino acids.RT-PCR results showed BcACT express in flower buds of both maintainer "Bajh97-05B’and Polima CMS line’Bpol97-05A’. Further studies showed that BcACT gene was an anther-specific one and up-regulated during anther development.(7)A gene,designated BcPRO(Brassica campestris profilin protein gene),was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF492988. The complete coding DNA sequence of BcPRO was 405 bp in length encoding 134 amino acids and shared more than 90%identity with those of B.napus and A.thaliana.RT-PCR showed that BcPRO gene expressed in stageⅢ,ⅣandⅤflower buds of maintainer and in stageⅢandⅣflower buds of Polima CMS. However,there was no detection in all tissues of Ogura CMS.RT-PCR revealed BcPRO gene expressed only in anthers,not in sepals,petals,filaments and pistils.These results suggested that BcPRO gene was an anther-specific one and might play important roles in pollen development.(8)Chalcone synthase(CHS)probe indicated different expression pattern among three Chinese cabbage-pak-choi materials.We designed gene-specific primer pairs according reported sequences of CHS and isolated BcCHS(Brassica campestris chalcone synthase gene)from mixed flower buds of male fertile line.The gene was designated BcCHS and submitted to NCBI under the accession number of EF101136. BcCHS gene is 1,236 bp in length with a sole 75 bp intron.The complete cds of BcCHS is 1,188 bp in length encoding 395 amino acids.BcCHS gene expressed in gradeⅣ,Ⅴand open flowers of ’Bcajh97-01B’ and ’Bcogu97-06A’.Further studies showed BcCHS express in anthers and petals.Additionally,a spontaneous white-flower mutant of Chinese cabbage-pak-choi was found in our test fields,and all the plant characters except flower color were identical with wild type ones.We isolated BcCHS-wf gene using the same primers and PCR procedure as cloning BcCHS.Comparison of the genomic sequences revealed two mutations in BcCHS-wf,both with A to G transitions,one at position +37 bp and the other at +970 bp.Both nucleotide substitutions occurred in AGA codes for arginine into GGA for glycin at residue +13 and into AGC coding for serine at residue +229,respectively.更多还原