油菜BnMKK4全长基因的克隆及表达分析

从‘陇油6号’油菜中克隆得到了一个新的BnMKK4基因的cDNA,全长1317bp,其中包括993bp的开放阅读框,159bp的5’非翻译区（5^1UTR）,165bp的3。非翻译区（3’UTR）。与拟南芥AtMKK4有很高的同源性,因此命名为BnMKK4（GenBank登录号JF268686）。该基因编码330个氨基酸的蛋白质,分子量36．5kDa,等电点为9．01。实时荧光定量PCR结果显示该基因表达受低温胁迫和盐胁迫的诱导,表明该基因在油菜适应低温胁迫和盐胁迫的过程中发挥作用。     

Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli

A genomic clone encoding a thiohydroximate S -glucosyltransferase ( S -GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S -GT gene were present in B. napus . The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli , and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S -GT activity detected from the recombinant protein confirmed that this clone was indeed a S -glucosyltransferase.     

cDNA cloning and characterization of a new stress-responsive gene BoRS1 from Brassica oleracea var. acephala

A new stress-responsive gene BoRS1 (GenBank accession No. AY373021 ) was isolated from Brassica oleracea var. acephala by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoRS1 was 2076 bp and contained a 1851 bp open reading frame (ORF) encoding 617 amino acids. Sequence analysis indicated that the deduced BoRS1 shared some identities with LTI65 , RD29A, RD29B and COR78 from Arabidopsis thaliana . Southern blot analysis of genomic DNA indicated that other related genes existed and there were two copies of BoRS1 in the genome of B. oleracea . Northern blot analysis revealed that BoRS1 was up-regulated by cold, mannitol, NaCl and abscisic acid (ABA). Expressional fluctuation of time course with ABA implied a two-step induction process. Tissue-specific expression analysis indicated that BoRS1 was expressed in all the tested plant tissues including leaves, stems and roots. Our studies imply that BoRS1 is a new gene that is responsive to environmental stresses such as low temperature, salinity and osmotic stress.     

油菜BnMKK2基因的克隆及表达分析

通过实验从陇油6号油菜中克隆得到了一种新的MAPK激酶基因BnMKK2基因的cDNA,全长1 344 bp,其中包括5′非翻译区（5′UTR）111 bp,3′非翻译区（3′UTR）165 bp,开放阅读框（ORF）长1 068 bp,编码355个氨基酸,该基因编码的蛋白质分子量为39.3 kDa,理论等电点为6.8。与拟南芥AtMKK2有很高的同源性,因此命名为BnMKK2(GenBank登录号:HQ848661)。该基因实时荧光定量PCR分析表明,BnMKK2基因的表达受低温胁迫诱导。     

非生物逆境胁迫下ZmCIPK10基因表达分析

干旱、高盐、低温、高温等非生物逆境严重影响植物的生长发育,研究参与逆境胁迫应答基因具有重要的理论意义和应用价值。从玉米中克隆了一个CIPK蛋白激酶基因,暂时命名为ZmCIPK10。该基因全长2 730 bp,转录长度2 100 bp,编码438个氨基酸。顺式元件分析发现在基因启动子区域存在ABRE、HSE、TC-rich repeats等推测的逆境顺势元件。荧光实时定量PCR结果表明ZmCIPK10在干旱、低温、盐胁迫下表达量上调,在ABA、高温胁迫下表达量下调。研究结果初步证实ZmCIPK10基因响应非生物逆境胁迫,为ZmCIPK10参与植物逆境信号途径及其功能研究提供理论依据。     

Isolation and characterization of a serine/threonine protein kinase SOS2 gene from Brassica napus

A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The full-length cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants.     

逆境胁迫对油菜谷胱甘肽还原酶基因表达及其酶活性的影响

利用cDNA末端快速分离(RACE)技术从陇油6号油菜中克隆得到一个新的谷胱甘肽还原酶基因GR2,全长2073 bp,开放阅读框1692 bp,编码563个氨基酸,预测蛋白质分子量为60.7 kDa,等电点7.9.实时荧光定量PCR分析表明:GR2基因在油菜根、茎、叶中均有表达,其中在叶中表达量最高.GR1和GR2基因的转录以及谷胱甘肽还原酶(GR)活性受到低温、高温、干旱、高盐胁迫的诱导,表明油菜谷胱甘肽还原酶在抵御低温、高温、干旱、高盐胁迫过程中发挥重要作用.脱落酸(ABA)预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显上升,表明ABA可以诱导GR1和GR2基因表达和GR酶活性.MAPKK抑制剂U0126预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显下降,表明U0126对GR1、GR2基因表达以及GR酶活性有抑制作用.     

Molecular cloning and characterization of a novel cold-regulated gene from Capsella bursa-pastoris.

A novel cor gene was cloned from Capsella bursa-pastoris (designated Cbcor15b) by RACE-PCR. The full-length cDNA of Cbcor15b was 652bp and contained a 417bp open reading frame (ORF) encoding a 139-amino acid hydrophilic protein. Multiple alignments showed that Cbcor15b had high similarity with other cold-regulated genes from Arabidopsis thaliana (cor15b, cor15a), Brassica napus (bn115, bn19 and bn26) and genes encoding late embryogenesis abundant (LEA) proteins. The predicted CbCOR15B protein was found to have a potential chloroplast signal sequence cleavage site, two cAMP- and cGMP-dependent protein kinase (PKA and PKG) phosphorylation sites. Cold acclimation assay showed that Cbcor15b was relevant to cold acclimation. Our study implies that Cbcor15b might have similar functions possessed by other cor genes in increasing plants' freezing tolerance.     

napin基因启动子克隆及进化分析

napin是一个重要的种子贮藏蛋白,它以组织特异性方式表达形成。以甘蓝型油菜为材料,克隆了napin基因启动子,测序结果表明,该启动子全长1 135bp,含有启动子特有的TATA-box等调控元件。系统进化分析显示,拟南芥、萝卜、白菜型油菜和甘蓝型油菜napin基因启动子之间既具有较高的同源性,又存在一定的差别。通过进化分析,指导我们在油菜脂肪酸改良过程中,采用不同的启动子在脂肪酸改良方面可能起到不同的效果。     

Molecular Cloning and Characterization of a Novel Dehydrin Gene from Ginkgo biloba

A full-length cDNA encoding a dehydrin was cloned from the living fossil plant Ginkgo biloba by rapid amplification of cDNA ends (RACE). The cDNA, designated as GbDHN, was 813 bp long containing an open reading frame of 489 bp. The deduced GbDHN protein had 163 amino acid residues, which formed a 17 kDa polypeptide with a predicted isoelectric point (pI) of 5.75. GbDHN had an S-segment and a K-segment, indicative of dehydrins, but no Y-segments. Homology analysis indicated that the S-segment and K-segment of GbDHN shared identity with those of other reported dehydrins, indicating that GbDHN belonged to dehydrin superfamily. Genomic sequence of GbDHN was also cloned using genomic walker technology. By comparing genomic DNA with the cDNA, it was found that there was a 257-bp intron in this gene. Promoter analysis indicated that it contained six CAAT boxes, one TATA box, one ABRE box and one GC-motif in the 5′-flanking region. Southern blot analysis revealed that GbDHN belonged to a single copy gene family. RT-PCR analysis revealed that GbDHN constitutively expressed in stems and roots. The increased expression of GbDHN was detected when G. biloba seedlings were treated with exogenous abscisic acid (ABA), salt stress and drought stress. These results indicate that the GbDHN has the potential to play a role in response to ABA and environmental stresses that can cause plant dehydration.     

柠条锦鸡儿肌动蛋白基因的克隆和表达稳定性分析

以几种豆科植物中肌动蛋白的氨基酸保守序列设计简并引物,采用RT—PCR结合RACE扩增技术,从柠条锦鸡儿叶片中克隆到一个编码肌动蛋白的基因,命名为CkACT。该基因cDNA全长为1655bp,开放阅读框1134bp,编码377个氨基酸。氨基酸比对分析表明,该基因编码的氨基酸与其他植物肌动蛋白基因具有较高的同源性。不同组织中的表达基本上一致,不同发育时期的基因表达相似,低温、NaCl、干旱和ABA处理后的表达强度没有明显差异,说明CkACT基因表达稳定。     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.