【作者】 李焰焰；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2008， 博士

【摘要】 十字花科蔬菜是我国栽培面积最大、总产量最高、杂种优势利用最为普遍的蔬菜之一。发掘和创建其雄性不育系在生产实践中具有重要意义,一直以来深受人们重视。但是,雄性不育的产生机制仍然是一个尚未完全解开的谜,目前对其的认识是零散和不完全的,许多问题还有待深入研究。花粉发育相关基因的克隆和分析有助于通过反义技术等阻断与花粉发育有关基因的表达从而获得雄性不育植株。这些分子生物学理论与技术的应用,不仅为最终阐明植物雄性不育机制提供了条件。也将大大推动十字花科作物和其他农作物杂种优势的有效应用。本实验室采用cDNA-AFLP技术从白菜(Brassica campestris L.ssp.chinensis Markino,syn.B.rapa ssp.chinensis)减数分裂胞质分裂突变体mmc(male meiotic cytokinesis)的野生型(可育株)中分离到两个与花粉育性密切相关的差异片段A8T4和A9T15,本研究以其为研究对象,通过PCR扩增方法对其基因组全长和cDNA全长进行克隆、鉴定,以及蛋白质结构和功能的预测,并以十字花科不同种属的植物为材料进行同源序列克隆、鉴定,并构建分子进化树,探讨其进化关系。并运用反义和RNA干涉技术,对菜心[B.campestris L.ssp.chinensis Markino var.parachinensis(Bailey)Tsen et Lee]进行转化,以获得这两个基因功能缺失突变体,从而分析其在花粉发育进程中的生物学功能。获得的主要结果如下:(1)根据差异片段A8T4和A9T15序列设计引物,利用RACE技术获得花粉发育相关基因的cDNA和DNA序列全长,分别命名为Brassica campestris Male Fertility 13(BcMF13)和Brussicacampestris Male Fertility 14(BcMF14)。序列分析表明,BcMF13的cDNA序列全长为600bp,含有2个外显子和1个长度为106bp的内含子。cDNA序列的最大开放阅读框(open reading frame,ORF)为222bp,编码73个氨基酸。在GenBank数据库中没有发现与BcMF13同源性很高的基因,表明BcMF13是一个新基因。BcMF13在可育株系‘Bajh97-01/B’的的Ⅳ～Ⅴ级花蕾中特异表达,尤其在雄蕊中表达量很高,在不育株系‘Bajh97-01A’中不表达,并且EST数据库中相似性很高的序列都来源于芸薹属的生殖器官,因此初步推断BcMF13很可能参与芸薹属花粉的发育过程。BcMF14的ORF为240个碱基,编码79个氨基酸,软件分析表明BcMF14的核酸序列和编码的BcMF14序列均符合RALF的特征,表明该基因是白菜中快速碱化因子类信号分子。(2)根据BcMF13的全长序列设计引物,在十字花科芸薹属植物的8个物种中克隆到BcMF13的同源序列,BcMF13同源基因在核苷酸水平和氨基酸水平相似性分别为80.0%～96.9%和96.7%～99.9%。在序列同源比对的基础上,基于Kimura双参数距离,构建了BcMF13同源基因在芸薹属植物8个物种中的NJ、ME和MP分子进化树,3种分子树的拓扑图总体趋势相同,有较高的一致性,表明用BcMF13序列得到的系统树能客观地反映BcMF13类基因在这些物种中的进化关系。(3)根据白菜BcMF14的全长序列设计引物,分别在十字花科5个属17份材料中扩增到BcMF14的同源序列,表明该基因在十字花科植物中广泛存在,进一步扩大了快速碱化因子的基因家族成员。并且这17个同源基因在核苷酸水平和氨基酸水平均有很高的相似性,编码的氨基酸都含有快速碱化因子蛋白的特征:一个保守的“YIXY”区和4个保守的半胱氨酸残基。说明该基因在十字花科植物中进化速度较慢,它们可能在其发育过程中发挥重要的作用。(4)利用BcMF13构建了含有组成型启动子CaMV35S的RNA反义载体pBI35S-AMF13和干涉载体pBI35S-RMF13,并利用农杆菌介导的方法将构建的BcMF13反义载体和干涉载体导入菜心中,分别获得了BcMF13的反义和干涉菜心转基因植株。利用BcMF14构建了含有组成型启动子CaMV35S的RNA反义载体pBI35S-AMF14和含有绒毡层特异表达启动子BcA9的RNA反义载体pBIBcA9-AMF14,并利用农杆菌介导的方法将构建的BcMF14的反义载体导入菜心中,获得了反义BcMF14菜心转基因植株。(5)用BcMF13 cDNA序列作探针的Northern杂交结果表明,BcMF13在含有CaMV35S启动子的反义载体和干涉载体的菜心转基因植株花蕾中的表达比对照都有降低,而干涉载体比反义载体对BcMF13表达水平的降低程度要大一些,说明干涉技术对白菜BcMF13的沉默效果要好于反义技术。透射电镜观察发现BcMF13受到反义和干涉抑制后,使花粉的发育在减数分裂前期就出现异常,在四分体时期小孢子和绒毡层细胞受到严重影响,从而导致正常的成熟花粉数量锐减。花粉萌发实验表明,pBI35S-AMF13菜心转基因植株花粉的萌发率为34.23%,显著低于对照的78.17%,而扫描电镜下的花粉畸形率达到54.66%。这种情况在转干涉基因的菜心植株中更明显:pBI35S-RMF13菜心转基因植株花粉的萌发率只有26.27%;花粉畸形率比转反义基因的稍高,达到57.55%。柱头萌发比体外萌发更能说明花粉正常与否,对受精后的柱头进行苯胺蓝染色观察发现转基因菜心的花粉管在柱头中的生长速度明显减慢。(6)用BcMF14 300-bp长的cDNA序列作探针,对菜心转基因植株大花蕾的Northern杂交结果表明,BcMF14在含有不同启动子反义载体的转基因菜心中的表达都受到不同程度的抑制,其中pBIBcA9-AMF14比pBI35S-AMF14的反义转基因植株中的表达量降低幅度更明显,说明BcA9启动子对BcMF14反义基因在花蕾中的启动效果要优于CaMV35S启动子。BcMF14的表达受到了抑制,一方面说明BcMF14为一在花粉中起作用的RALF基因,另一方面也验证了CaMV35S启动子在菜心花粉中具有启动活性。在对菜心转BcMF14反义基因植株和转空载体对照植株的细胞学观察和透射电镜的观察中均发现,转基因植株与对照株在四分体时期以前的小孢子发育阶段没有明显差别,而两种转基因菜心花粉在发育到四分体阶段都出现异常,说明BcMF14很可能作用在白菜小孢子发育的四分体时期。BcMF14除了直接影响小孢子发育以外,还对绒毡层的发育产生了影响。推测BcMF14基因在小孢子和绒毡层之间的信号、物质联络中有一定的作用。pBI35S-AMF14、pBIBcA9-AMF14反义载体菜心转基因植株花粉在扫描电镜下的花粉畸形率分别为48.95%和51.2%,花粉的体外萌发率分别为44.39%和26.09%,花粉在柱头上的萌发和对照相比,只有部分花粉管能生长到达花柱底部,这表明反义技术使得BcMF14的表达量降低,导致花粉发育异常,产生大量畸形花粉,最终导到花粉萌发率下降。这表明快速碱化因子类基因家族中的确存在一些调控植物花粉发育的RALF信号分子。这与Bedinger等对在番茄花粉中特异表达的PRALF基因进行研究结果相吻合,PRALF或BcMF14的过量或缺失表达都能引起花粉萌发异常,进而影响花粉管的生长。更多还原

【Abstract】 Cruciferae crops is one of the vegetable crops species of the highest yield and cultivated most widely as well as is a kind of crops of the most successful in utilizing of heterosis in China. In recent years, an increasingly amount of research has been devoted to the molecular biology of research on the plant breeding of the male sterile line and the basis for application in Brassica crops. However, the mechanism of the plant male sterility is not demonstrated adequately. A lot of questions remain unknown. The cloning and characterization of pollen development related genes is important to analysis the mechanism of male sterility in Cruciferae and construct male sterility line. Along with the development of molecular biological theories and methods, it can provide powerful tools to study on the molecular mechanism of plant male sterility and drive the heterosis to apply in the production in Brassica crops and others. Recently, by cDNA-AFLP technology, two differential fragments named A8T4 and A9T15, 191 bp and 201 bp respectively in length, were obtained in our lab from the wild type of male meiotic cytokinesis (mmc) mutant (male sterile plant) of Brassica campestris L. ssp. chinensis Markino (syn. B. rapa ssp. chinensis). The genes which the two fragments belonging to were named BcMF13 and BcMF14. Earlier study had proved that BcMF13 was a new gene while BcMF14 shared a high homology to Rapid Alkalinization Factor (RALF) genes. In this study, the full length cDNA and DNA of pollen development related BcMF13 and BcMF14 gene was cloned and characterized from Chinese cabbage pak-choi (B. campestris L. ssp. chinensis Makino). Homologous genes with the BcMF13 and BcMF14 were cloned from deferent species in deferent genera of Cruciferae by PCR amplification. Phylogenetic trees of BcMF13 and BcMF14 gene were constructed based on the alignment of their homologous genes, respectively. They provided important molecular data for the study of Cruciferae evolution. Meanwhile, RNAi or anti-sense RNA technology were applied to transforme B. campestris L. ssp. chinensis Markino var. parachinensis (Bailey) Tsen et Lee by agrobacterium-mediated method to obtain their loss-of-function mutant to analyze BcMF13 and BcMF14 function in pollen development process. The major study results were as follows:(1) On the basis of cDNA-AFLP differential fragments A8T4 and A9T15, the cDNA sequence of BcMF13 and BcMF14 were isolated by rapid amplification of cDNA ends (RACE) . Subsequently, their DNA sequences were obtained. Sequence comparison indicated that the BcMF13 cDNA was 600 bp and was interrupted by a 106-bp intron. The largest open reading frame (ORF) of BcMF13 was composed of 222 bp with a deduced 73 amino acids. Homology analysis showed that BcMF13 shared no similarity with any known genes. So BcMF13 was a new gene. BcMF13 exclusively expressed in stage four and five flower buds, open flowers and silique of fertile line, strongly expressed in stamens with no expression in sterility. And many cDNA sequences from the ESTs share high similarity with BcMF13 all came from the generative organs in Brassica. So we suggested that BcMF13 most possibly had a role in pollen development of Brassica. ORF of BcMF14 was composed of 240 bp with a deduced 79 amino acids. Homology comparison and phylogenetic analysis showed that BcMF14 had relation to rapid alkalinization factor gene (RALF). The BcMF14 amino acids had the characteristic of RALF proteins.(2) BcMF13 Homologous genes were isolated from 8 species of Cruciferae by PCR amplification. The identification ratio of nucli acid and amino acid were 80.0% ~ 96.9% and 96.7% ~ 99.9%.The Clustal X program was used to align the homologous sequences from different 8 species of Cruciferae and to produce NJ, ME and MP phylogentic trees based on distances estimated by the two-parameter method with ] 000 bootstrap samples using MEGA 2;1. The result shows three kinds of phylogentic trees have the strictly accordant topologic structure which means the BcMF13 molecular tree can reflect the evolution of BcMF13.(3) BcMF14 Homologous genes were isolated from 17 species of Cruciferae by PCR amplification. The increased the RALF gene families into some new species. The entification ratio of nucli acid and amino acid were high. The proteins they coded all had the characteristic of RALF: one conserved ’YIXY’ region and four conserved C residues. These indicated the BcMF14 evolution was slow and it maybe has important role in Cruciferae.(4) The antisense expression vector and RNAi expression vector of BcMF13 containing constitutive promoter CaMV35S were constructed, respectively, named pBI35S-AMF13 and pBI35S-RMF13. By agrobacterium-mediated method, their flowering Chinese cabbage transgenic plantlets were obtained. The antisense expression vector of BcMF14 containing constitutive promoter CaMV35S and tapetum-expressed promoter BcA9 were constructed, respectively, named pBI35S-AMF14 and pBIBcA9-AMF14. By agrobacterium-mediated method, their flowering Chinese cabbage transgenic plantlets were obtained.(5) Using cDNA of BcMF13 as probe, the Northern hybridization results showed that the expression of BcMF13 in pBI35S-AMF13 and pBI35S-RMF13 transforments was sharply inhibited. And the expression of BcMF13 in pBI35S-RMF13 transforments was much lower than the pBI35S-AMF13 transforments. It suggested that the RNA interference technology was better than antisense technology on the BcMF13 gene in Brassica. Investigation of pollen morphology of transgenic plants of flower Chinese cabbage obtained from BcMF14 anti-sense RNA vector by transition electron microscope indicated that when BcMF13 was depressed pollen development got abnormal early in meiosis. The microspore and tapetum were affected. All these greatly reduced the number of mature pollens. As far as pBI35S-AMF13 was concerned, 34.23% pollen germination ratio in vitro and 54.66% pollen abnormality by scan electron microscope. This is more serious in the RNA interference technology, with 26.27% pollen germination ratio in vitro and 57.55% pollen abnormality in the pBI35S-RMF13. Whether pollen germination was normal or not should be confirmed by further in pistil experiment. Moreover, pollen germination in stained pistil by aniline blue after pollination 24 h by fluorescence microscope was examined. The results also indicate that the expression of BcMF13 was inhibited by the two methods, which resulted in adhesion failure of pollen to pistil and germination ration of pollen accordingly decreased.(6) Using 300-bp cDNA of BcMF14 as probe, the Northern hybridization was performed in flower buds of pBI35S-AMF14 and pBIBcA9-AMF14. The result showed that the expression of BcMF14 was caught and decreased sharply in comparison to positive CK, and the expression in pBIBcA9-AMF14 was more decreased than in pBI35S-AMF14, which proved that BcMF14 was a pollen-expressed RALF. This illuminated CaMV35S promoter had activity in pollen, and the activity was less than BcA9, which was consistent with the study results of our lab’s transition electron microscope experiments and tissue investigation showed that BcMF14. worked during tetrad. It worked on microspore and tapetum and might have a role in the interaction between the two kinds of cells. In pBI35S-AMF14,48.95% pollen germination ratio in vitro and 26.09% pollen abnormality by scan electron microscope also confirmed that CaMV35S promoter acted in pollen, which was 51.2% and 44.39% in pBIBcA9-AMF14. Moreover, pollen germination in pistil was examined. The results also indicate that less pollen tube could get into the bottom of the pistil on contrast to the control. This tells us that the antisense technology could decrease the expression of BcMF14 and made pollen development abnormal, with many freaky pollens and the co-identity between the pollen and the pistil was affected. All these indicated that in the RALF families there were some signal molecule had certain role in pollen development. These inosculated with Bedinger’s discovering, the over-expression or depress of BcMF14 and PRALF could disturb pollen development and restrain the pollen tubes’ growing remarkably.更多还原