Cloning and expressional characterization of soybean GmLls1 gene

In higher plants, green leaf is a very important part involved in the photosynthesis process. Good develop-ment of the leaves is the guarantee of high yields and quality. Leaf senescence that occurs in the last stage of leaf development is a genetically p…     

大豆叶片衰老过程中半胱氨酸蛋白酶基因的特异性表达

用反转录和多聚酶链反应（ＲＴ－ＰＣＲ）和琼脂糖凝胶电泳方法对大豆叶片衰老过程中半胱氨酸蛋白酶基因的表达情况进行了研究，发现由ＰＣＲ扩增出的ｃＤＮＡ带谱会随着叶片的不断衰老而发生较明显的改变，初步证明，至少有一条ｃＤＮＡ带为绿叶样品所特有，另外，两条ｃＤＮＡ带则为衰老叶片样品所特有，该实验结果为进一步分离大豆叶片衰老时特异表达基因及其启动子和最终实施转基因工程奠定了基础。     

与大豆叶片衰老相关的cDNA的克隆

植物叶片衰老是一个受遗传控制的细胞降解过程,最终导致叶片的死亡。一些研究结果已表明,蛋白酶基因的表达与叶片衰老过程相关,其中一些基因的表达具有衰老特异性。以半胱氨酸蛋白基因特异性引物和寡聚ｄＴ为引物,用ＲＴ－ＰＣＲ方法分别扩增来源于绿叶和诱导衰老叶片ｍＲＮＡ的互补ＤＮＡ（ｃＤＮＡ）,然后进行琼脂糖凝胶电泳,差异显示出一条衰老叶片样品所特有的ｃＤＮＡ带,将此ｃＤＮＡ进行了克隆和序列分析。分析结果证明     

白桦转BpGH3.5基因叶片早衰突变株的光合特性及生长分析

【目的】植物叶片早衰将影响作物产量和品质,研究叶片早衰机制对于培育耐早衰优良品种具有重要意义。【方法】以转BpGH3.5基因的白桦叶片早衰突变株(G4)、非转基因白桦(WT)及叶片正常的转基因白桦(G21)等为材料,测定其叶绿素含量、净光合速率(P_n)、气孔导度(G_s)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)等光合指标,测定苗高的时序变化规律。【结果】叶片早衰突变影响了叶片叶绿素的合成及积累,突变株的叶绿素相对含量(SPAD)均值为36.08,相对两个对照株系分别下降7.34%、7.48%。叶片早衰突变影响了白桦光合呼吸作用。突变株的净光合速率、气孔导度、蒸腾速率分别为WT野生株系的67.54%、64.44%、64.93%,胞间CO₂浓度达到234.33 μmol/mol,显著高于G21对照株系( P<0.05)。突变株的当年高生长显著低于WT、G21 两个对照株系,当年高生长分别是WT、G21 两个对照株系的68.9%、85.0%。利用Logistic方程对3个参试株系当年苗高生长量的变化过程进行了拟合,其系数均高于0.98,同时,通过Logistic方程计算的生长参数揭示了早衰突变株高生长较两个对照株系低的原因是速生期苗高平均生长量(GR)、苗高日生长量的平均值(GD)等生长参数较低。【结论】转BpGH3.5基因的白桦发生了叶片早衰现象,使叶绿素提前降解,影响了光合呼吸作用,进而影响了苗高生长。     

Molecular characterization of GmNHX2, a Na＋/H＋ antiporter gene homolog from soybean,and its heterolosous expression to improve salt tolerance in Arabidopsis

Na＋/H＋ antiporters have been well documented to enhance plant salt tolerance by regulating cellular ion homeostasis. Here, a putative Na＋/H＋ antiporter gene homolog GmNHX2 from soybean was cloned and predicted to encode a protein of 534 amino acids with 10 putative transmembrane domains. GmNHX2 was expressed in all soybean plant tissues but enriched in roots and its expression was induced by NaCI and polyethylene glycol （PEG） treatments. GmNHX2 exhibits greater sequence similarity with LeNHX2 and AtNHX6 than that of AtNHX1 and AtSOS1. Although phylogenetic analysis clustered GmNHX2 with organellar （tonoplast and vesicles） antiporters, the GmNHX2-EGFP （enhanced green fluorescent protein） fusion protein was possibly localized in the plasma membrane or organelle membrane of transgenic plant cells, Furthermore, transgenic Arabidopsis plants expressing GmNHX2 were more tolerant to high NaCl concentrations during germination and seedling stages when compared with wild-type plants. These results suggest that GmNHX2 is a membrane Na＋/H＋ antiporter and may function to regulate ion homeostasis under salt stress.     

Generation of selectable marker-free transgenic rice resistant to chewing insects using two co-transformation systems

To produce selectable marker-free (SMF) transgenic rice resistant to chewing insects, the Bacillus thuringiensis cryIA(c) gene (Bt) was introduced into two elite japonica rice varieties by using two Agrobacterium-mediated co-transformation systems. One system is with a single mini-twin T-DNA binary vector in one Agrobacterium strain, which consists of two separate T-DNA regions, one carrying the Bt while the other contains the selectable marker gene, hygromycin resistant gene (HPT). The other system uses two separate binary vectors in two separate Agrobacterium cultures, containing the Bt or HPT gene on individual plasmids. A lot of independent transgenic rice lines harboring both Bt and selectable marker genes were obtained. The results showed that the co-transformation frequency of the Bt gene and HPT gene was much higher by using the mini-twin T-DNA vector system (29.87%) than that by the two separate binary vector systems (4.52%). However, the frequency of the SMF transgenic rice plants obtained from the offspring of co-transgenic plants (21.74%) was lower for the mini-twin T-DNA vector system than that for the latter (50-60%). The data of ELISA implied that the expressed Bt proteins were quantitated as 0.025-0.103% of total leaf soluble proteins in the transgenic plant. Therefore, several elite transgenic rice lines, free of the selectable marker gene, were chosen. The results from both in vitro and in vivo insect bioassays indicated that the SMF transgenic rice was shown to be highly resistant to the striped stem borer and rice leaf folder. Moreover, in a natural field condition without any insecticide applied, all the transgenic rice plants were found to be not injured by the rice leaf folder, whereas the wild types were impaired seriously.     

转AlNHX1基因大豆后代遗传稳定性及耐盐性分析

为了获得耐盐性有所提高的转AlNHX1基因大豆后代材料,以已获得的转AlNHX1基因的6个株系中的3个株系后代为研究对象,通过分别对这3个株系转基因大豆各后代进行PCR分子检测并结合耐盐性鉴定,以分析外源基因在转基因大豆中遗传稳定性和耐盐性.结果表明:AlNHX1基因在转基因植株的后代中遗传;选取3个株系中部分阳性植株做耐盐性检测,结果表明:转基因大豆耐盐性状均好于野生型大豆.在150mmol/L NaCl胁迫下,转基因大豆叶片中维持了相对较高的K+/Na+比值,相对含水量较野生型提高了9%,而渗透势降低了39%,表明转基因大豆具有较好的吸水和保水能力;在盐胁迫下,超氧化物歧化酶(SOD)与过氧化物酶(POD)活性较野生型大豆分别提高了45%与69%.综上,通过耐盐筛选获得的转AlNHX1基因大豆具有较强的耐盐性.     

大豆叶片57kD钙依赖蛋白激酶自磷酸化性质的研究

本实验证明大豆叶片质膜上57kD钙依赖蛋白激酶具有较强的体外自磷酸化活性；并且其体外自磷酸化水平受到人工诱导衰老处理的明显促进及外源6－BA预处理的有效抑制，暗示该激酶可能参与外源细胞分裂素对大豆叶片衰老的调控过程。进一步的生化分析表明，其自磷酸化位点可能发生在丝氨酸和苏氨酸等残基上，而在酷氨残基上未发生磷酸化反应。     

Genetic analysis and gene mapping of a dominant presenescing leaf gene <Emphasis Type="Italic">PSL3</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Leaf senescence as an active process is essential for plant survival and reproduction. However, premature senility is harmful to agricultural production. In this study, a rice mutant, named as psl3 (presescing leaf 3) isolated from EMS-treated Jinhui 10, displays obvious premature senility features both in morphological and physiological level. Genetic analysis showed that mutant trait was controlled by a single dominant gene (PSL3), which was located on rice chromosome 7 between SSR marker c7sr1 and InDel marker ID10 with an interval of 53.5 kb. The result may be useful for the isolation of the PSL3 gene.     

水稻OsNAP基因的功能分析及其在育种价值方面的研究

NAC转录因子是植物特异的转录因子.本研究通过从水稻日本晴(Oryza sativa L.)的基因组中克隆到一个与拟南芥衰老相关的基因AtNAP(Atlg69490)的同源基因OsNAP.OsNAP的ORF为1 179bp,推测其编码蛋白含有392个氨基酸,等电点为8.55,分子质量为42.195ku.为进一步研究OsNAP的功能,利用显性抑制元件SRDX构建其pCAMBIA1304-35S:OsNAP-SRDX表达载体,通过农杆菌浸染方法将其转化水稻中花11品种.T1代转基因植株通过潮霉素、RealtimePCR、和Western blot鉴定,筛选出4个OsNAP高表达的阳性植株.在种子成熟期,观察到T1代转基因4号植株较野生型相比有明显的延缓衰老的表型,其他农艺性状如单株分蘖数、单株总粒数、单穗饱和穗粒数、千粒重、结实率等都优于野生型.实验证明抑制OsNAP基因的表达能够延缓水稻叶片衰老并可以提高产量,具有潜在的育种价值.     

Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

Structural and biochemical mechanism responsible for the stay-green phenotype in common wheat

In order to increase the supply of assimilated carbon to grain, a new stay-green wheat cultivar, Chuannong17 (CN17), with delayed leaf senescence, carrying wheat-rye 1RS.1BL translo- cated chromosome was developed. CN17 exhibited distinct differences in net photosynthetic rate (Pn), chlorophyll (Chl) content, malondialdehyde (MDA) content, activity of both superoxide dismutase (SOD) and catalase (CAT) during the grain filling stage, and flag leaf senescence compared with the control. The new cultivar maintained longer and higher photosyn- thetic competence compared with the control, and this aspect correlated with the difference in chloro-plast development. Moreover, the stay-green pheno-type of CN17 was also observed under natural growth conditions. Consequently, the coordination of the physiological, biochemical, and structural aspects in the stay-green cultivar produced higher seed weights and per-plant yield compared with the control cultivar.     

丙型肝炎病毒E2基因在转基因番茄植株中的表达

以T-E1E2为模板扩增得到丙型肝炎病毒包膜蛋白基因E2,构建该基因的植物表达载体p35s-E2．通过农杆菌介导的叶盘法转化番茄子叶．转基因番茄植株叶片总DNA的PCR、Southern blot检测结果表明,E2基因已整合进了转基因番茄植株基因组中;RT-PCR,Western blot分析证实E2基因在转基因番茄植株叶片中表达．     

邻单胞菌邻苯二酚1,2-双加氧酶基因(tfdC)在拟南芥中表达的初步研究

将从一株邻单胞菌中克隆到的一个新的邻苯二酚1，2－双加氧酶基因（tfd C)的起始密码子由GTG突变成ATG，并克隆到农杆菌双元载体pPZPY122中，利用农杆菌介导转化模式植物拟南芥，获得了转化植株，经过PCR，PCR－Southern和Southern dot blot方法检测证实，ftd C基因已经整合到拟南芥基因组中，邻苯二酚1，2－双加氧酶酶活性检测表明，转基因植株具有一定的酶活性，而未转化的植株则不具有酶活性。     

抗盐基因Gm01g04890大豆子叶节遗传转化研究

利用根癌农杆菌介导的大豆子叶节转化方法,将转录因子HD-Zip家族基因Gm01g04890分别导入测序品种Willimas 82(W82)和小粒豆品种东农50(DN50)两种大豆品种的子叶节中.探讨了种子萌发时间、农杆菌菌液浓度、侵染条件、共培养时间和乙酰丁香酮(AS)浓度等因素对大豆子叶节形成不定芽的影响,优化了大豆子叶节遗传转化体系.T0代再生植株经草铵膦筛选以及RT-PCR检测,Gm01g04890基因已成功转入并整合于大豆基因组,获得了转Gm01g04890基因的DN50大豆抗性植株.     

春小麦不同叶位及叶龄叶片若干生理生化指标的动态变化

对春小麦开花后的不同中位及叶龄叶片在衰老过程中的相关生理生化指标进行了分析。结果表明：不同叶位叶片，在同一生育期内，旗叶的蛋白质含量，叶绿素含量和ＣＡＴ活性均高于其他叶位叶片，ＭＤＡ含量和外渗电导率值低于其他叶位叶片，随着叶龄的增加，旗叶的这些生理生化指标的变化趋势都慢于其他叶位叶片。