野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料,采用同源克隆方法和RT-PCR技术获得一个TIFY类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明,与其他物种相比,GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高,达到56%;序列分析表明GsTIFY11b蛋白除具有TIFY保守结构域外,还具有一个N端保守结构域和一个C端保守的Jas结构域;实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达;将GsTIFY11b转化拟南芥来验证其耐盐碱功能,获得两个转基因纯合体株系,盐碱胁迫分析结果表明,GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性,并且与野生型相比,转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明,GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明,GsTIFY11b定位于细胞核中。上述结果表明,该基因在细胞核中起着转录调节子的作用,可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料, 采用同源克隆方法和RT-PCR技术获得一个TIFY 类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明, 与其他物种相比, GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高, 达到56%; 序列分析表明GsTIFY11b蛋白除具有 TIFY保守结构域外, 还具有一个N端保守结构域和一个C端保守的Jas结构域; 实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达; 将GsTIFY11b转化拟南芥来验证其耐盐碱功能, 获得两个转基因纯合体株系, 盐碱胁迫分析结果表明, GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性, 并且与野生型相比, 转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明, GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明, GsTIFY11b定位于细胞核中。上述结果表明, 该基因在细胞核中起着转录调节子的作用, 可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

Coordinated activation of metabolic pathways for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively termed jasmonates, are ubiquitous plant signalling compounds. Several types of stress conditions, such as wounding and pathogen infection, cause endogenous JA accumulation and the expression of jasmonate-responsive genes. Although jasmonates are important signalling components for the stress response in plants, the mechanism by which jasmonate signalling contributes to stress tolerance has not been clearly defined. A comprehensive analysis of jasmonate-regulated metabolic pathways in Arabidopsis was performed using cDNA macroarrays containing 13516 expressed sequence tags (ESTs) covering 8384 loci. The results showed that jasmonates activate the coordinated gene expression of factors involved in nine metabolic pathways belonging to two functionally related groups: (i) ascorbate and glutathione metabolic pathways, which are important in defence responses to oxidative stress, and (ii) biosynthesis of indole glucosinolate, which is a defence compound occurring in the Brassicaceae family. We confirmed that JA induces the accumulation of ascorbate, glutathione and cysteine and increases the activity of dehydroascorbate reductase, an enzyme in the ascorbate recycling pathway. These antioxidant metabolic pathways are known to be activated under oxidative stress conditions. Ozone (O3) exposure, a representative oxidative stress, is known to cause activation of antioxidant metabolism. We showed that O3 exposure caused the induction of several genes involved in antioxidant metabolism in the wild type. However, in jasmonate-deficient Arabidopsis 12-oxophytodienoate reductase 3 (opr3) mutants, the induction of antioxidant genes was abolished. Compared with the wild type, opr3 mutants were more sensitive to O3 exposure. These results suggest that the coordinated activation of the metabolic pathways mediated by jasmonates provides resistance to environmental stresses.     

Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.     

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.     

GsSLAH3, a Glycine soja slow type anion channel homolog,positively modulates plant bicarbonate stress tolerance

Alkaline stress is a major form of abiotic stress that severely inhibits plant growth and development, thus restricting crop productivity. However, little is known about how plants respond to alkali. In this study, a slow‐type anion channel homolog 3 gene, GsSLAH3 , was isolated and functionally characterized. Bioinformatics analysis showed that the GsSLAH3 protein contains 10 transmembrane helices. Consistently, GsSLAH3 was found to locate on plasma membrane by transient expression in onion epidermal cells. In wild soybeans, GsSLAH3 expression was induced by NaHCO₃ treatment, suggesting its involvement in plant response to alkaline stress. Ectopic expression of GsSLAH3 in yeast increased sensitivity to alkali treatment. Dramatically, overexpression of GsSLAH3 in Arabidopsis thaliana enhanced alkaline tolerance during the germination, seedling and adult stages. More interestingly, we found that transgenic lines also improved plant tolerance to KHCO₃ rather than high pH treatment. A nitrate content analysis of Arabidopsis shoots showed that GsSLAH3 overexpressing lines accumulated more NO₃^? than wild‐type. In summary, our data suggest that GsSLAH3 is a positive alkali responsive gene that increases bicarbonate resistance specifically.     

Global analysis of non-coding small RNAs in Arabidopsis in response to jasmonate treatment by deep sequencing technology

In plants, non-coding small RNAs play a vital role in plant development and stress responses. To explore the possible role of non-coding small RNAs in the regulation of the jasmonate (JA) pathway, we compared the non-coding small RNAs between the JA-deficient aos mutant and the JA-treated wild type Arabidopsis via high-throughput sequencing. Thirty new miRNAs and 27 new miRNA candidates were identified through bioinformatics approach. Forty-nine known miRNAs (belonging to 24 families), 15 new miRNAs and new miRNA candidates (belonging to 11 families) and 3 tasiRNA families were induced by JA, whereas 1 new miRNA, 1 tasiRNA family and 22 known miRNAs (belonging to 9 families) were repressed by JA.     

HOS1, a genetic locus involved in cold-responsive gene expression in arabidopsis.

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19 degrees C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10 degrees C or higher. Compared with the wild type, hos1-1 plants are l ess cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.     

小拟南芥ApZFP基因异源超表达促进拟南芥开花并提高耐逆性

锌指蛋白(ZFP)是一类重要的转录因子, 广泛参与植物的生长发育和非生物胁迫应答。新疆小拟南芥(Arabidopsispumila)又名无苞芥, 是十字花科短命植物, 具有高光效、繁殖力强和适应干旱等生物学特征, 而且比模式植物拟南芥(A.thaliana)更耐高盐胁迫。将前期克隆的小拟南芥锌指蛋白基因ApZFP通过花滴法转化到哥伦比亚生态型拟南芥(Col-0)中,获得了独立表达的转基因株系。表型观察发现, 过量表达ApZFP基因可促使拟南芥在长短日照下均提前开花。实时荧光定量PCR结果显示, 转基因拟南芥株系中, 光周期途径中的CO基因和年龄途径中的SPL基因表达上调; 春化、环境温度和自主途径中的FLC基因表达下调; 编码成花素的基因FT及下游开花相关基因AP1和LFY的表达量均升高。进一步通过盐、干旱和ABA胁迫处理ApZFP转基因株系的种子和幼苗, 发现在胁迫处理下, 与对照相比, 转基因拟南芥种子萌发率较高, 幼苗主根较长。因此推测, ApZFP在植物发育过程中具有多种功能, 可能既参与植物的开花转变过程, 又同其它植物的锌指蛋白基因一样, 参与植物的耐逆过程。