拟南芥抗菌核病突变体275-7的初步研究

草酸是核盘菌致病过程中产生的毒素因子。以菌核病菌毒素草酸（3 mmol/L）为筛选压,从1 000个拟南芥T-DNA插入突变体中筛选出了1个草酸不敏感突变体,命名为275-7。通过拟南芥活体接种对突变体275-7进行菌核病抗性鉴定,与野生型相比,突变体275-7对菌核病的抗性显著增强（P<0.05）;荧光定量PCR检测结果表明,275-7中茉莉酸途径的标志基因PDF1.2的表达量是野生型的12倍,而水杨酸途径的标志基因PR1基因的表达量与野生型比较无差异。推测拟南芥突变体275-7可能通过增强水杨酸介导的防卫反应,从而表现出对菌核病的抗性。     

拟南芥CYP76C2基因(At2g45570)的克隆及其菌核病抗性功能分析

以拟南芥基因组DNA为模板,扩增出细胞色素P450(CYP76C2)基因的ORF,成功构建了CYP76C2基因的过表达载体并获得转基因植株.通过离体和活体接种表明,CYP76C2基因的过表达植株对核盘菌的抗性增强.     

枯草芽孢杆菌草酸脱羧酶转化拟南芥研究初报

多种真菌在致病过程中会分泌草酸,这些草酸产生菌寄主范围广,可以导致上百种植物病害,造成世界范围农作物的严重减产。草酸可以通过氧化与脱羧两种途径降解。该研究克隆了枯草芽孢杆菌的草酸脱羧酶基因Yvrk,并构建其植物表达载体,通过农杆菌浸花转化拟南芥,收获种子,用除草剂Basta筛选获得15株转基因植株,对其中的11个株系分别离体接种菌核病菌,24h后量病斑,发现有6株转基因植株的病斑显著小于野生型。PCR验证发现大部分转基因植株确有Yvrk的存在,实验结果说明草酸脱羧酶基因确能一定程度上缓解真菌病害。     

HIGS-SsCCS转基因拟南芥的菌核病抗性鉴定

【目的】菌核病是由核盘菌(Sclerotinia sclerotiorum)引起的一类真菌病害,核盘菌寄主范围广泛,严重危害多种作物的品质。本研究利用寄主诱导基因沉默(HIGS)的方法在寄主中诱导核盘菌致病相关基因的沉默从而增强寄主的菌核病抗性,为菌核病抗病育种提供新的思路。【方法】铜锌超氧化物歧化酶是一种重要的抗氧化剂,以核盘菌铜锌超氧化物歧化酶铜伴侣基因(copper chaperone for copper/zinc superoxide dismutase,SsCCS)为靶基因,通过生物信息学分析该基因的结构特点,并利用MEGA6.0软件构建系统发育树;通过分别比对拟南芥及核盘菌基因组,选择特异的干扰片段进行扩增;采用农杆菌介导的浸花序法,将HIGS载体转入拟南芥Col-0,通过DNA鉴定以及标记筛选出稳定的HIGS-CCS转基因拟南芥;选取4—5周龄的HIGS-CCS转基因拟南芥植株叶片接种核盘菌野生菌株1980,于接种24 h后统计病斑面积,分析转基因株系的菌核病抗性;通过qRT-PCR分析核盘菌侵染转基因植株过程中SsCCS的表达情况;同时在接种6、12、24 h后利用D...     

Transformation of Arabidopsis thaliana via Agrobacterium tumefacience with an endochitinase gene from Trichoderma, and enhanced resistance to Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Trichoderma endochitinase gene ThEn-42 at initial bud stage. Eleven seedlings (corresponding to about 0.22 percent transformation) exhibited resistance to hygromycin. The DNA fragment unique to endochitinase (ThEn-42) was amplified by Arabidopsis leaf-PCR or genomic DN…     

The Physiological and Molecular Responses of Arabidopsis thaliana to the Stress of Oxalic Acid

Many fungal phytopathogens can secrete oxalic acid （OA）, which is the crucial pathogenic determinant and plays important roles in pathogenicity and virulence of pathogen during infection process. However, how plants respond to OA stress still needs further characterization. In this study, we observed the physiological and molecular responses of Arabidopsis thaliana to OA stress. The leaves of 6-wk-old A. thaliana were sprayed with OA and distilled water respectively, and 0, 2, 4, 8, 12, and 24 h later, the leaves were collected and the contents of MDA, H2O2, and GSH, and the activities of CAT, SOD, and POD were determined and the expressions of PR1 and PDF1.2 were also studied. Under the stress of 30 mmol L-1 OA, SOD activity was first enhanced to reduce the accumulation of O2.-. But immediately, POD, CAT, and GSH all decreased extremely resulting in the accumulation of H2O2, and the MDA content increased 24 h later. GSH activity was enhanced significantly at 24 h after OA used. However, H2O2 wasn＇t eliminated at the same time, suggesting that the activity inhibitions of POD and CAT might be the reasons that caused Arabidopsis cells＇ impairment under OA stress. RT-PCR results indicated that PDF1.2, a marker gene of the JA/ET signaling was significantly induced; PR1, an indicator gene in SA signaling, was slighlty induced from 8 to 12 h after OA stress. In conclusion, Arabidopsis may recruit metabolism of reactive oxygen, both JA/ET and SA signaling pathways to respond to OA stress. These results will facilitate our further understanding the mechanisms of plant response to OA and OA-dependent fungal infection.     

拟南芥钴胺素合成相关蛋白CSRP基因克隆及其功能分析

以拟南芥为研究对象,根据已知的生物信息数据,通过反向遗传学的方法,对与钴胺素合成相关蛋白CSRP基因进行分子克隆,得到该基因的基因组序列和cDNA序列,构建二元重组质粒,利用根癌农杆菌介导的花浸泡转基因方法分别转化突变体和野生型植株,进行互补试验和过表达试验,筛选转基因植株,并进一步预测其编码蛋白的亚细胞定位于叶绿体中.结果表明,在突变体中转入该基因后,转基因植株恢复野生型的表型,而在过表达的转基因植株中表型更为明显,即生长旺盛,晚花.说明该基因对于拟南芥的营养生长起促进作用,由于其功能预测与钴胺素合成相关的蛋白,推测其可能在植物营养生长中起作用.     

叶片喷施C1化合物对拟南芥生理特性和C1代谢、光合作用及胁迫相关基因表达的影响

为了比较甲醇、甲醛和甲酸3种C1化合物对植物生理特性及基因表达的影响,更好地理解这3种C1化合物的作用机制,分别用5 mmol· L-1甲醇、甲醛、甲酸溶液均匀喷洒于温室条件下盆栽培养的拟南芥叶片上,以喷蒸馏水的植物为对照,每周喷1次,4周后分析施用这3种C1化合物对其生长及生理特性的影响.结果表明:甲醇促进拟南芥生长,而甲醛和甲酸抑制其生长,甲醛的抑制作用特别明显;甲醇处理显著降低了叶片花青素含量但提高了叶绿素a含量及叶绿素a/b的比值,甲醛处理显著降低了叶绿素a含量及叶绿素a/b的比值,甲酸处理显著提高了叶绿素a含量但大幅度降低了叶绿素b含量从而使叶绿素a/b的比值比对照高2倍;3种C1化合物处理都能增加叶片中可溶性总蛋白质含量,甲醇处理尤为明显,只有甲醛处理可显著提高叶片可溶性总糖、H2O2及羰基化蛋白质含量,产生氧化胁迫.对基因表达谱进行逆转录聚合酶链式反应(RT-PCR)的结果表明:所选的大多数光合作用相关基因的表达被甲醇诱导,而甲醛抑制它们的表达但诱导很多胁迫相关基因的表达,甲酸对大多数光合作用相关基因的表这没有显著影响;3种C1化合物对大多数C1代谢相关基因没有显著影响,但都抑制5,10-亚甲基-四氢叶酸还原酶基因的表达.