紫花苜蓿盐诱导类受体蛋白激酶基因MsSIK1的克隆及功能分析

【目的】对紫花苜蓿（Medicago sativa L. cv. Zhongmu-1）stress-induced protein kinase gene 1（MsSIK1）进行克隆与表达研究,了解该基因的分子机制及其应用。【方法】以紫花苜蓿叶片总RNA为模板,根据同源克隆设计简并引物,利用RT-PCR结合RACE技术,获得MsSIK1的编码序列。利用同源性比对进行序列分析。通过SMART网站（http://smart.embl-heidelberg.de/）模拟该基因的蛋白结构。构建MsSIK1的亚细胞定位瞬时表达载体,使用基因枪转化法将MsSIK1与GFP在洋葱表皮细胞中融合瞬时表达并观察其亚细胞定位荧光信号。通过Real time-PCR分析MsSIK1在NaCl、ABA和干旱处理条件下的表达特征。利用农杆菌侵染方法获得转基因拟南芥植株,通过RT-PCR对转基因植株进行表达鉴定,获得转基因植株后,利用转基因株系进行盐处理进而对成苗期转基因拟南芥性状鉴定。在盐胁迫处理下,测定野生型与转基因株系的叶绿素含量、MDA含量进而验证该基因的抗盐功能。【结果】获得MsSIK1编码序列2 478 bp,编码825个氨基酸。该蛋白C端与多种植物激酶具有相当高的同源性,模拟蛋白结构发现该基因具有类受体蛋白激酶高度保守的丝氨酸/苏氨酸结构域、跨膜结构域和富含亮氨酸重复序列的膜外结构域。Real time-PCR分析表明该基因在NaCl、ABA和干旱处理条件下上调表达,其中在盐处理条件下,MsSIK1表达先升高后降低,在处理4 h时达到最大值（约为对照值的7倍）。在干旱胁迫处理时,MsSIK1受诱导表达增强明显,当处理2 h时表达量达到最大值（约为对照值的6倍）;ABA处理时,MsSIK1被诱导表达明显,当处理3 h时表达量达到最大值,约为对照值的6.8倍。MsSIK1与GFP融合瞬时表达的洋葱表皮细胞中的荧光信号主要集中于质膜附近,转化空载体的洋葱表皮细胞中的荧光信号分布于细胞各个部位。转基因植株的RT-PCR鉴定表明,T₁代6个株系中所得到的MsSIK1条带明显、亮度高,且T₁-10中表达量最高;但在野生型中检测不到该条带,说明外源基因已经整合到拟南芥染色体中并能遗传到子代。成苗期转基因拟南芥盐处理后发现T₃-2、T₃-6、T₃-10转基因株系较野生型植株长势好,说明MsSIK1的转入提高了拟南芥的抗盐性。与对照相比,转MsSIK1拟南芥在NaCl处理下,叶绿素含量下降较少,其中,野生型叶绿素含量降低了77%,T₃-3降低了53%,T₃-6降低了44%,T₃-10降低了35%;同样盐胁迫下,3个转基因株系的MDA含量积累较少,其中,野生型MDA的含量是T₃-10株系的1.3倍。【结论】MsSIK1作为一个类受体蛋白激酶受多种逆境胁迫诱导,该基因的过量表达提高了拟南芥的抗盐性。     

毛果杨Eukaryotic translation initiation factor 5A（eIF5A） 同源基因的生物信息学分析

为研究毛果杨eIF5A基因间的同源性及其进化关系,以毛果杨的eIF5A基因为研究对象,利用生物信息学软件及网站对其进行碱基分布、氨基酸组成、亲疏水性、保守区以及二级结构和三级结构的预测与分析,并与其他物种的eIF5A氨基酸序列进行多重比对与进化分析。结果表明,4个毛果杨eIF5A基因定位于不同染色体上,且都只含有5个外显子;研究还发现不同成员间氨基酸数目、氨基酸序列间的疏水性存在一定的差异;亚细胞定位分析表明,4个eIF5A蛋白均定位于细胞质上;二级结构预测结果显示,4个eIF5A氨基酸序列以无规则卷曲、扩展链和α-螺旋为主要组成部分,且4条氨基酸序列三维结构十分相似。上述结果均为毛果杨eIF5A基因家族的进一步功能分析提供了一定基础。     

The chemical treatments combined with antagonistic yeast control anthracnose and maintain the quality of postharvest mango fruit

During the storage and transportation of the mango fruit, the major source of disease is anthracnose, caused by the fungus Colletotrichum gloeosporioides. The objective of this study is to find an appropriate method that not only reduces mango decay but also maintains its postharvest quality. The effects of chemicals, the use of the yeast species Metschnikowia pulcherrima and their combination on storage quality, focusing on the enzyme activity related to disease of Tainong mangos was studied. By immersing the mangoes in M. pulcherrima suspension(1.0×10~8 cfu mL~(–1)), salicylic acid(SA) solution(50 mg L~(–1)) or calcium chloride(CaCl_2) solution(1.0 g L~(–1)), the lesion expansion and decay of the mango fruit caused by C. gloeosporioides could be significantly delayed. These treatments also delayed the changes in quality traits(a~* value, firmness, contents of total soluble solids(TSS) and vitamin C(Vc), while the activities of anti-disease enzymes such as polyphenol oxidase(PPO), phenylalanine ammonia lyase(PAL), chitinase(CHT) and β-1,3-glucanase(GUN) were enhanced as compared to the control treatment. Furthermore, the combination of SA solution, CaCl_2 solution and M. pulcherrima suspension presented an additive effect, increasing the efficacy in controlling the disease and maintaining the storage quality of mango fruits. Taken together, our data suggest that the integration of chemical treatments combined with M. pulcherrima could be an alternative to the use of fungicides in postharvest treatment of the mango fruit, specifically for improving storage quality as well as the control of the anthracnose.     

Up-regulation of a homeodomain-leucine zipper gene HD-1 contributes to trichome initiation and development in cotton

Plant trichomes originate from epidermal cells. In this work, we demonstrated that a homeodomain-leucine zipper (HD-Zip) gene, Gh_A06G1283 (GhHD-1A), was related to the leaf trichome trait in allotetraploid cotton and could be a candidate gene for the T₁ locus. The ortholog of GhHD-1A in the hairless accession Gossypium barbadense cv. Hai7124 was interrupted by a long terminal repeat (LTR) retrotransposon, while GhHD-1A worked well in the hairy accession Gossypium hirsutum acc. T586. Sequence and phylogenetic analysis showed that GhHD-1A belonged to the HD-Zip IV gene family, which mainly regulated epidermis hair development in plants. Silencing of GhHD-1A and its homoeologs GhHD-1D in allotetraploid T586 and Hai7124 could significantly reduce the density of leaf hairs and affect the expression levels of other genes related to leaf trichome formation. Further analysis found that GhHD-1A mainly regulated trichome initiation on the upper epidermal hairs of leaves in cotton, while the up-regulated expression of GhHD-1A in different organs/tissues also altered epidermal trichome development. This study not only helps to unravel the important roles of GhHD-1A in regulating trichome initiation in cotton, but also provides a reference for exploring the different forms of trichome development in plants.     

过量表达蔗糖转运蛋白基因增强转基因小麦的耐旱性

【目的】创制过量表达TaSUT1A的转基因小麦,分析TaSUT1A在转基因小麦中的遗传及其对干旱胁迫的应答反应,选育抗旱的转基因小麦新种质。【方法】采用基因重组技术构建了TaSUT1A表达载体,利用基因枪介导法将该载体转入小麦品种科农199,通过Bialaphos筛选、转化植株基因组DNA PCR验证获得转基因T₀植株;利用RT-PCR检测TaSUT1A在转基因T₃植株中的表达情况,在此基础上,对3个转基因系的T₄转基因植株进行抗旱性鉴定和抗旱相关生理指标分析,验证其抗旱能力。【结果】经PCR检测和RT-PCR验证,获得了转TaSUT1A小麦阳性植株,与非转基因对照相比,20%PEG胁迫处理显著诱导了转基因株系根叶组织中目标基因TaSUT1A的上调表达。抗旱鉴定和抗性生理分析显示,在20%PEG胁迫处理下,转基因株系的萌发率比非转基因对照平均提高了32.8%,显著高于非转基因对照,并促进初生根的萌发和生长,初生根长和胚芽鞘长比非转基因对照平均增加了81.72%和170.77%;在20%PEG胁迫处理下,转基因植株叶组织中的蔗糖和可溶性糖平均提高了42.95%和36.56%,根中蔗糖和可溶性糖平均提高了58.01%和43.01%,均显著高于非转基因对照植株;与未胁迫处理相比,20%PEG胁迫处理后非转基因植株叶中的SOD活性由105.4 U·g^-1FW升高到139.1 U·g^-1FW,而转基因植株的活性由107.7-115.3 U·g^-1FW提高到168.2-211.6 U·g^-1FW,显著高于非转基因对照,同时,转基因小麦株系的MDA的产生较非转基因对照平均降低了37.47%,显著减少了MDA的产生。【结论】TaSUT1A在参与植物的逆境应答反应机制中具有重要作用,促进逆境胁迫中小麦的萌发和生长,超量表达TaSUT1A可显著提高转基因小麦的耐旱能力。     

Genome-wide identification and characterization of the JAZ gene family and its expression patterns under various abiotic stresses in Sorghum bicolor

The jasmonate ZIM domain (JAZ) protein belongs to the TIFY ((TIF[F/Y]XG) domain protein) family, which is composed of several plant-specific proteins that play important roles in plant growth, development, and defense responses. However, the mechanism of the sorghum JAZ family in response to abiotic stress remains unclear. In the present study, a total of 17 JAZ genes were identified in sorghum using a Hidden Markov Model search. In addition, real-time quantification polymerase chain reaction (RT-qPCR) was used to analyze the gene expression patterns under abiotic stress. Based on phylogenetic tree analysis, the sorghum JAZ proteins were mainly divided into nine subfamilies. A promoter analysis revealed that the SbJAZ family contains diverse types of promoter cis-acting elements, indicating that JAZ proteins function in multiple pathways upon stress stimulation in plants. According to RT-qPCR, SbJAZ gene expression is tissue-specific. Additionally, under cold, hot, polyethylene glycol, jasmonic acid, abscisic acid, and gibberellin treatments, the expression patterns of SbJAZ genes were distinctly different, indicating that the expression of SbJAZ genes may be coordinated with different stresses. Furthermore, the overexpression of SbJAZ1 in Escherichia coli was found to promote the growth of recombinant cells under abiotic stresses, such as PEG 6000, NaCl, and 40°C treatments. Altogether, our findings help us to better understand the potential molecular mechanisms of the SbJAZ family in sorghum in response to abiotic stresses.     

SlGH9-15 regulates tomato fruit cracking with hormonal and abiotic stress responsiveness cis-elements

Fruit cracking occurs easily during the late period of fruit development when plants encounter an unsuitable environment, dramatically affecting fruit production and marketing. This study conducted the bulked segregant RNA-Seq (BSR) to identify the key regulatory gene of fruit cracking in tomatoes. BSR-Seq analysis illustrated that two regions associated with irregularly cracking were located on chromosomes 9 and 11, containing 127 candidate genes. Further, through differentially expression analysis and qRT-PCR in cracking-susceptible and cracking-resistant genotypes, the candidate gene SlGH9-15 (Solyc09g010210) with significantly differential expression levels was screened. Bioinformatics analysis of the GH9 gene family revealed that 20 SlGH9 genes were divided into three groups. The phylogenetic analysis showed that SlGH9-15 was closely related to cell wall construction-associated genes AtGH9B1, AtGH9B6, OsGH9B1, and OsGH9B3. The cis-acting elements analysis revealed that SlGH9-15 was activated by various hormones (ethylene and ABA) and abiotic stresses. The expression pattern indicated that 13 SlGH9 genes, especially SlGH9-15, were highly expressed in the cracking-susceptible genotype. Its expression level gradually increased during fruit development and achieved maximum value at the red ripe stage. Additionally, the cracking-susceptible tomato showed higher cellulase activity and lower cellulose content than the cracking-resistant tomato, particularly at the red ripe stage. This study identified SlGH9-15 as a key gene associated with fruit cracking in tomatoes for the first time and gives new insights for understanding the molecular mechanism and complex regulatory network of fruit cracking.     

Cloning and Characterization of a Salt Tolerance-Associated Gene Encoding Trehalose-6-Phosphate Synthase in Sweetpotato

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes： trehalose-6-phosphate synthase（TPS） and trehalose-6-phosphate phosphatase（TPP）. In the present study, a TPS gene, named IbTPS, was first isolated from sweetpotato（Ipomoea batatas（L.） Lam.） cv. Lushu 3 by rapid amplification of cDNA ends（RACE）. The open reading frame（ORF） contained 2 580 nucleotides encoding 859 amino acids with a molecular weight of 97.433 kDa and an isoelectric point（pI） of 5.7. The deduced amino acid sequence showed high identities with TPS of other plants. Real-time quantitative PCR analysis revealed that the expression level of IbTPS gene was significantly higher in stems of Lushu 3 than in its leaves and roots. Subcellular localization analysis in onion epidermal cells indicated that IbTPS gene was located in the nucleus. Transgenic tobacco（cv. Wisconsin 38） plants over-expressing IbTPS gene exhibited significantly higher salt tolerance compared with the control plant. Trehalose and proline content was found to be significantly more accumulated in transgenic tobacco plants than in the wild-type and several stress tolerance related genes were up-regulated. These results suggest that IbTPS gene may enhance salt tolerance of plants by increasing the amount of treahalose and proline and regulating the expression of stress tolerance related genes.     

Overexpression of AmDUF1517 enhanced tolerance to salinity,drought, and cold stress in transgenic cotton

As abiotic stresses become more severe as a result of global climate changes, the growth and development of plants are restricted. In the development of agricultural crops with greater stress tolerance, AmDUF1517 had been isolated from the highly stress-tolerant shrub Ammopiptanthus mongolicus, and can significantly enhance stress tolerance when inserted in Arabidopsis thaliana. In this study, we inserted this gene into cotton to analyze its potential for conferring stress tolerance. Two independent transgenic cotton lines were used. Southern blot analyses indicated that AmDUF1517 was integrated into the cotton genome. Physiological analysis demonstrated that AmDUF1517-transgenic cotton had stronger resistance than the control when treated with salt, drought, and cold stresses. Further analysis showed that trans-AmDUF1517 cotton displayed significantly higher antioxidant enzyme (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione S-transferase (GST)) activity and less reactive oxygen species (ROS) accumulation, which suggests that overexpression of AmDUF1517 can improve cotton resistance to stress by maintaining ROS homeostasis, as well as by alleviating cell membrane injury. These results imply that AmDUF1517 is a candidate gene in improving cotton resistance to abiotic stress.     

苹果异三聚体G蛋白α亚基基因MdGPA1的克隆及功能鉴定

【目的】异三聚体G蛋白(Heterotrimeric G protein)作为植物生物体内重要的信号转导分子,在感受外界环境刺激、参与植物抗逆反应和跨膜信号转导等方面发挥着重要作用。克隆异三聚体G蛋白α亚基基因MdGPA1,并在烟草中过量表达MdGPA1,对其进行生物学功能鉴定和生理指标分析,为多年生木本植物响应环境因子信号转导过程中的分子机理研究提供参考。【方法】本研究以‘嘎拉’苹果(Malus×domestica‘Royal Gala’)为研究试材,利用同源序列比对和PCR技术,克隆获得MdGPA1。使用MEGA5.0构建GPA1物种间系统进化树;利用qRT-PCR方法检测该基因在苹果受非生物胁迫诱导表达及组织特异性表达情况。构建MdGPA1植物过表达载体,通过农杆菌介导法转化烟草叶片,比较干旱胁迫条件下野生型和转基因株系的表型与生理指标,验证MdGPA1在植物干旱胁迫条件下的生物学功能。【结果】克隆得到苹果异三聚体G蛋白α亚基基因MdGPA1(基因序列号:MDP0000881842),该基因长为1 173 bp,编码390个氨基酸。进化树分析表明MdGPA1与白梨Pb GPA1亲缘关系最近,同源性最高。基因表达分析显示MdGPA1主要在叶片中表达,在根系中的表达量次之,在茎和果实中的表达量较低。定量分析表明,该基因参与干旱、低温和盐等非生物逆境胁迫响应,在150 mmol·L~(-1)Na Cl、150 mmol·L~(-1)甘露醇、10%PEG和4℃胁迫条件下表达量明显下调,在5%H_2O_2胁迫处理下表达量明显上调。在烟草中过量表达MdGPA1,发现MdGPA1转基因烟草表现出对干旱敏感的表型特征,其叶片鲜重、叶绿素含量以及脯氨酸含量明显低于野生型烟草。在地下部,MdGPA1转基因烟草同样表现出对干旱敏感的表型特征;其根系形态相比于野生型较小,干重也明显低于野生型。【结论】MdGPA1参与了植物感受外界环境刺激的过程,对干旱、低温和盐等非生物逆境胁迫都存在着不同程度的响应。在烟草中异源表达MdGPA1后,提高了烟草对干旱的敏感性,转基因烟草表现出不耐干旱的表型,受干旱胁迫比野生型烟草更为严重,说明MdGPA1在响应植物抗旱胁迫中起着负调控作用。     

牦牛应激型 HSPA1A和 HSPA2基因的克隆及序列分析

为了解牦牛应激型 HSPA1A和 HSPA2基因的特点,根据GenBank已公布的牛应激型 HSPA1A和 HSPA2基因序列设计4对引物,每个基因分两段扩增,测序并拼接,首次克隆牦牛应激型 HSPA1A和 HSPA2基因。序列分析表明,扩增到的牦牛应激型 HSPA1A基因全长2 134 bp,开放阅读框全长1 926 bp,编码641个氨基酸,分子质量为70.26 ku; HSPA2基因全长1 911 bp,开放阅读框全长1 911 bp,编码636个氨基酸,分子质量为69.85 ku。将 HSPA1A和 HSPA2基因开放阅读框编码的氨基酸序列进行ScanProsite分析,均得到3个HSP70蛋白家族标记。核苷酸序列同源性分析表明, HSPA1A和 HSPA2基因具有较高的保守性,牦牛与牛的 HSPA1A和 HSPA2基因核苷酸序列同源性最高,分别为99.8%和99.1%。遗传进化关系分析表明,牦牛与牛的应激型 HSPA1A和 HSPA2基因亲缘关系最近。     

Cloning and Expression Analysis of Sucrose Non-fermenting-1 Related Protein Kinase （SnRK） in Cucumis sativus L. Under Low Nitrogen Conditions

The sucrose non-fermenting-1 related protein kinase（SnRK）, whose expression is induced by kinds of hyperosmotic stresses, plays a key role in improving stress resistance of plants. In order to investigate the molecular mechanism of low nitrogen resistance in cucumber, the full-length cDNA of SnRK gene was cloned in this study. The result showed that SnRK gene was 1 548 bp in length, encoded 515 amino acids, and had more than 80% homology with other crops. The protein encoded by this gene was an unstable and hydrophilic protein with no transmembrane structure and no signal peptide. Under nitrogen-free conditions and low nitrogen conditions, the expression pattern analysis of SnRK gene showed that this gene was up-regulated and its expression increased and was significantly higher than the normal level as the nitrogen concentration decreased. In addition, the expression of SnRK gene was also inhibited in the high nitrogen level and was significantly lower than the normal level. The result of this study would help us understand the molecular mechanism of low nitrogen resistance in cucumber.