白菜雄性不育相关基因BcMF4基因功能的RNAi验证

BcMF4(Brassica campestris Male Fertility 4)是前一阶段从普通白菜 (Brassica campestris ssp. chinensis var. communis, syn. B. rapa ssp. chinensis var. communis)核雄性不育两用系的可育株中分离到的雄性不育相关基因。本研究根据BcMF4基因的cDNA序列设计两对特异引物, 从普通白菜花蕾cDNA中扩增出两个片断后连接至双元载体pBI12l中, 得到了RNAi (RNA interference) 植物表达栽体pBI-B4R, 并导入了农杆菌LBA4404菌株中; 通过组织培养途径转化菜心(B. campestris ssp. chinensis var. parachinensis), 72.2%的菜心转基因植株中45.8%的花粉为缩小而空瘪的畸形, 而且这些植株的花粉离体萌发率降低至23.7%; Northern杂交显示, 转基因植株的花粉畸形, 是由于BcMF4基因片段的插入使BcMF4基因的表达受到了抑制。结果表明, 采用RNAi技术下调了BcMF4基因的表达, 导致了菜心转基因植株部分花粉的不育, 证明BcMF4基因在普通白菜和菜心等白菜植物的花粉发育中起着重要作用。     

Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis

The gene Brassica campestris male fertility 13 (BcMF13, GenBank accession number EF158459) was isolated as a reproductive organ-specific gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). It is exclusively expressed in stage four and five flower buds of fertile lines and is most strongly expressed in stamens. Here, we report a functional characterization of this BcMF13 gene in the antisense-silenced plants. The inflorescence of the BcMF13 mutant was compacted with anthers curved outside. The fertility of this mutant was greatly reduced with less than 5 seeds per silique. Under scanning electron microscopy, the mutant demonstrated numerous shriveled pollen grains with deep invaginations. The frequency of normal pollen grains was just 45.34%. The pollen mother cell, the tetrad, and the mature pollen of the BcMF13 mutant were abnormal resulting in the poor pollen vitality. Germination test in vivo suggested BcMF13 delayed the pollen tubes’ extension in the style. All these indicated BcMF13 had a vital role in pollen development of Chinese cabbage.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi (Brassica campestris L. ssp. chinensis Makino)

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function,whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG)gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

花粉发育相关基因BcMF13不同转录本的克隆及分析

在克隆花粉发育相关基因BcMF13时,用RACE技术进行3’端扩增,得到了两个不同长度的3’转录本。与EST库比对,发现与其相似性高的序列都来源于十字花科芸薹属、萝卜属等植物的生殖器官,并且在这些近源属中也出现不同转录本。这些不同转录本的存在说明BcMF13存在剪切异构体,调控过程灵活复杂,反映出该基因具有重要功能。同时,对BcMF13基因的RT-PCR表达研究表明,它只在可育株系中表达,在不育株系中不表达,且主要集中在大花蕾、雄蕊中表达。分析BcMF13推导的蛋白结构,发现其编码的蛋白包含多个生物活性位点。以上结果均表明BcMF13与花粉育性相关,多个剪切异构体的发现说明BcMF13在执行生理功能时较为活跃。     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

雄性不育相关基因msLTP的反义RNA验证

根据普通白菜雄性不育相关的脂质转移蛋白基因(msLTP)的cDNA序列设计引物,从普通白菜花蕾的cDNA中扩增出312bp的片段,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中,通过农杆菌介导法转化菜心;利用PCR和Southern blot分析检测得到了25株转基因植株,转基因植株的花粉部分畸形或空瘪,花粉离体萌发率为38.56%,较未转化植株的萌发率(76.32%)降低了37.76个百分点.研究表明,反义RNA技术使msLTP基因沉默而导致了菜心转基因植株的部分花粉发育不良,说明msLTP基因在普通白菜和菜心等花粉发育中具有重要作用.