拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花

AtLH基因是BcpLH基因在拟南芥(Arapsis thaliana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域.在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关.为研究AtLH的基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥.与野生型比较页言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量的植株.这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腑处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强,转基因植株对激素的敏感性改变,IAA剌激根生长的作用增强,ABA抑制根生长的作用减弱.由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲.     

大白菜BcpLH基因的原核表达及其蛋白产物的Western分析

BcpLH gene preferentially expressed in folding leaf of Chinese cabbage contains dsRNA-binding domains. The cDNA of BcpLH gene was cloned into a His-fusion expression vector pET-28a (+) and was induced to express in E. coli strain BL21 (DE3). Then, the specific protein was partially purified and the rabbit was immunized to prepare the anti-serum. Meanwhile BcpLH cDNA was cloned into the pMAL-c2 containing the solubizing partner, and then the soluble protein generated. It was demonstrated from Western dot assay that the BcpLH protein was specific. The BcpLH active protein and its anti-serum made it possible to study RNA-binding activity and regulation mechanism in plant development.     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花(英文)

AtLH基因是BcpLH基因在拟南芥(Arabidopsis thealiana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域。在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关。为研究AtLH基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥。与野生型比较而言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量表达的植株。这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腋处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强;转基因植株对激素的敏感性改变,IAA刺激根生长的作用增强,ABA抑制根生长的作用减弱。由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲。     

大白菜BcPLH基因的原核表达及其蛋白产物的Western分析

BcpLH基因是大白菜包叶组织特异的新基因,含有双链RNA结合域。在含有His标记序列的原核表达载体pET28-a(+)上插入Bc-pLH基因的cDNA,在大肠杆菌BL21(DE3)中诱导表达出了特异性蛋白,并免疫大白兔制备出高效价的抗血清;同时,将BcpLH基因插入到含有超级助溶剂的pMAL-c2载体上,并在大肠杆菌DH5α中诱导表达,结果获得了可溶的蛋白。Western斑点印迹分析的结果证明了BcpLH的特异性。BcpLH活性蛋白及其抗血清的产生为研究BcpLH基因的RNA结合     

Leafy head formation of the progenies of transgenic plants of Chinese cabbage with exogenous auxin genes

The experiment was performed to evaluate the progenies of plant lines transgenic for auxin synthesis genes derived from Ri T-DNA.Four lines of the transgenic plants were self-crossed and the foreign auxin genes in plants of T5 generation were confirmed by Southern hybridization.Two lines,D1232 and D1653,showed earlier folding of expanding leaves than untransformed line and therefore had early initiation of leafy head.Leaf cuttings derived from plant of transgenic line D1653 produced more adventitious roots than the control whereas the cuttings from folding leaves had much more roots than rosette leaves at folding stage,and the cuttings from head leaves had more roots than rosette leaves at heading stage.It is demonstrated that early folding of transgenic leaf may be caused by the relatively higher concentration of auxin.These plant lines with auxin transgenes can be used for the study of hormonal regulation in differentiation and development of plant orgens and for the breeding of new variety with rapid growth trait.     

水稻双链RNA结合蛋白同源基因OsRBP的克隆及其表达的分析

在基因数据库中发现两个水稻EST片段与大白菜BcpLH基因的双链RNA结合结构域 (dsRBD)有同源的区域 ,根据同源片段的位置特征设计引物 ,用RT-RCR扩增粳稻 (Oryzasativasubsp .japonica)愈伤组织的cDNA ,得到大小为 1.8kb的DNA片段。该cDNA片段含完整的编码区 ,有两个典型的dsRBD ,分别与BcpLH的dsRBD在氨基酸水平上同源性为 75 %左右 ,故将其命名为OsRBP。RT -PCR表达分析显示该基因在未成熟的种子和愈伤组织中表达 ,在根、茎、叶、穗、成熟种子及胚芽鞘中没有表达信号 ,由此推测该基因的表达可能与种子和胚的早期发育相关。该研究首次从水稻中分离到双链RNA结合蛋白基因 ,并初步研究了其表达方式 ,为进一步探讨水稻重要器官的发育和植物中双链RNA结合蛋白的调节作用奠定了基础     

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.     

BrpSPL9 (Brassica rapa ssp. pekinensis SPL9) controls the earliness of heading time in Chinese cabbage

The leafy heads of cabbage (Brassica oleracea), Chinese cabbage (Brassica rapa ssp. pekinensis), Brussels sprouts (B. oleracea ssp. gemmifera) and lettuce (Lactuca sativa) comprise extremely incurved leaves that are edible vegetable products. The heading time is important for high quality and yield of these crops. Here, we report that BrpSPL9‐2 (B. rapa ssp. pekinensis SQUAMOSA PROMOTER BINDING‐LIKE 9‐2), a target gene of microRNA brp‐miR156, controls the heading time of Chinese cabbage. Quantitative measurements of leaf shapes, sizes, colour and curvature indicated that heading is a late adult phase of vegetative growth. During the vegetative period, miR156 levels gradually decreased from the seedling stage to the heading one, whereas BrpSPL9‐2 and BrpSPL15‐1 mRNAs increased progressively and reached the highest levels at the heading stage. Overexpression of a mutated miR156‐resistant form of BrpSPL9‐2 caused the significant earliness of heading, concurrent with shortening of the seedling and rosette stages. By contrast, overexpression of miR156 delayed the folding time, concomitant with prolongation of the seedling and rosette stages. Morphological analysis reveals that the significant earliness of heading in the transgenic plants overexpressing BrpSPL9‐2 gene was produced because the juvenile phase was absent and the early adult phase shortened, whereas the significant delay of folding in the transgenic plants overexpressing Brp‐MIR156a was due to prolongation of the juvenile and early adult phases. Thus, miR156 and BrpSPL9 genes are potentially important for genetic improvement of earliness of Chinese cabbage and other crops.     

Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage

An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The N-terminal amino acid sequence of the protein was highly homologous to that of plant cyclophilins and consequently the protein was denoted as C-CyP. To understand the antifungal activity of C-CyP, we isolated a cDNA encoding its gene from a Chinese cabbage leaf cDNA library. The Chinese cabbage genome bears more than one C-CyP gene copy and C-CyP mRNA is highly expressed in all tissues except the seeds. Recombinant C-CyP catalyzed the cis-trans inter-conversion of the Ala-Pro bond of the substrate, which indicates this protein has peptidyl-prolyl cis-trans isomerase activity. It also inhibited the growth of several fungal pathogens.     

Characterization of a salicylic acid- and pathogen-induced lipase-like gene in Chinese cabbage

A cDNA clone for a salicylic acid-induced gene in Chinese cabbage (Brassica rapa subsp. pekinensis) was isolated and characterized. The cabbage gene, designated Br-sil1 (for Brassica rapa salicylate-induced lipase-like 1 gene), encodes a putative lipase that has the family II lipase motif GDSxxDxG around the active site serine. A database search showed that plant genomes have a large number of genes that contain the family II lipase motif. The lipase-like proteins include a myrosinase-associated protein, an anther-specific proline-rich protein APG, a pollen coat protein EXL, and an early nodule-specific protein. The Br-sil1 gene is strongly induced by salicylic acid and a nonhost pathogen, Pseudomonas syringae pv. tomato, that elicits a hypersensitive response in Chinese cabbage. Treatment of the cabbage leaves with BTH, methyl jasmonate, or ethephon showed that the Br-sil1 gene expression is induced by BTH, but not by methyl jasmonate or ethylene. This indicates that the cabbage gene is activated via a salicylic acid-dependent signaling pathway. An examination of the tissue-specific expression revealed that the induction of the Br-sil1 gene expression by BTH occurs in leaves and stems, but not in roots and flowers. Without the BTH treatment, however, the Br-sil1 gene is not expressed in any of the tissues that were examined.     

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理, 将甘蓝型油菜(Brassica napus L.)和白菜(B. campestris L. ssp. chinensis Makino)杂交并连续回交6代获得白菜OguCMS, 在与保持系花药细胞学比较的基础上, 运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片断, 利用RACE克隆得到该片断的cDNA全长, 命名为BcMYBogu(GenBank 登录号: EF127861), 对其氨基酸序列和表达特征进行研究。结果表明, 白菜OguCMS绒毡层在四分体后增生, 高度液泡化, 导致小孢子花粉外壁异常, 细胞质同外壁分离并降解; 花药变白; BcMYBogu具有典型的MYB DNA结合域—W残基和SH[AL]QKY[RF]基序; 系统进化分析显示BcMYBogu与AtMYB26, AtMYB32和AtMYB4等聚类在同一分枝; RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达, 但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。     

不结球白菜BcLRK01基因对芜菁花叶病毒及胁迫诱导的响应

通过cDNA-AFLP技术,从芜菁花叶病毒(TuMV)侵染的不结球白菜幼叶中分离到一条差异表达的基因片段,克隆获得其cDNA全长为2 124bp,编码707个氨基酸的富亮氨酸重复类受体激酶,命名为BcLRK01。利用实时定量PCR研究了该基因在TuMV侵染及高盐、冷胁迫、水杨酸(SA)、茉莉酸(JA)、乙烯(ET)等处理下的表达情况,结果显示,TuMV侵染、高盐、冷胁迫、SA、JA和ET等均能诱导BcLRK01不同程度的表达,说明该基因可能是不结球白菜病毒病的病程相关基因,同时也参与高盐和冷胁迫以及SA、JA、ET等的信号途径。     

Molecular cloning and characterization of ascorbate oxidase gene in non-heading Chinese cabbage

The cDNA clone of ascorbate oxidase gene was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino, cv. Suzhouqing) and characterized. Sequence analysis showed that there was a high similarity between this sequence (named BcAO) and its homologues in other plant species. Southern blotting indicated that more than one nuclear gene encoded this enzyme in non-heading Chinese cabbage. The mRNA level of the BcAO gene in leaves was monitored by real-time PCR at different developmental stages and under different stress conditions. Results showed that the expression of BcAO was upregulated by light, and the BcAO gene responded to copper stress as well. After inoculation with Alternaria brassicae, the expression of BcAO in the leaves was increased in general and peaked at 12 and 72 h post inoculation, with much higher expression at the later date. Cloning the BcAO gene will enable us to further understand its function and would provide useful information for resistance breeding program for non-heading Chinese cabbage.