根癌农杆菌介导的水稻纹枯病菌转化系统的建立

为建立水稻纹枯病菌的T-DNA插入诱变转化系统,以水稻纹枯病菌(Rhizoctonia solani AG-1ⅠA)强致病力菌株GD118为转化的初始菌株,从预诱导时间、共培养时间、共培养时乙酰丁香酮的浓度、共培养温度和共培养时固体诱导培养基(SIM)的pH值等5个方面对转化条件进行了优化,成功地建立了适合于水稻纹枯病菌根癌农杆菌介导转化(ATMT)的优化系统。这个优化系统的转化条件如下:以30μg/mL的潮霉素B作为转化子的筛选浓度,预诱导8h,共培养20h,共培养时固体诱导培养基上的乙酰丁香酮浓度为200μmol/L,共培养温度25℃,共培养时固体诱导培养基pH5.6～5.8。采用这个系统筛选到的转化子继代培养5代后,在含30μg/mL潮霉素B的PDA平板上仍表现明显的抗性。从得到的转化子中随机抽取10个,利用根据抗潮霉素hph基因设计的特异性引物进行PCR扩增,转化子均能扩增出500bp左右的预期条带;与此同时,以4个根癌农杆菌作为阳性对照,用根癌农杆菌Vir基因特异引物对转化子进行PCR扩增,以排除转化子受农杆菌污染所致的假阳性,结果表明:4个根癌农杆菌均能扩增出Vir基因条带(730bp),而10个转化子均未能扩增出相应条带。以上两个PCR扩增的实验结果清楚地表明,T-DNA已经插入到目标菌株GD118中。     

胶孢炭疽菌T-DNA插入突变体库的构建及其分子分析

利用根癌农杆菌介导的T-DNA插入遗传转化体系转化杞果炭疽菌SC2菌株,共获得抗潮霉素B突变体4 680个,平均每转化1.0×106个炭疽菌分生孢子可得到300～980个突变体,且潮霉素抗性在多次传代实验中得到稳定遗传.随机挑取38个突变体进行PCR检测,均可扩增出1条约1.2 kb的目标谱带,说明突变体为T-DNA插入引起:进一步的Southern blot杂交分析结果表明,在随机挑选的20个突变体中,有1个(5%)为三拷贝T-DNA插入,4个(20%)为双拷贝插入,剩余的15个(75%)为单拷贝插入.     

农杆菌介导油菜黑胫病菌遗传转化

为优化建立农杆菌介导的油菜黑胫病菌遗传转化技术体系,以携带潮霉素磷酸转移酶基因的pCH-sGFP为载体,农杆菌LBA4404为介体,对油菜黑胫病菌的分生孢子进行转化.油菜黑胫病菌的最佳转化体系为:分生孢子浓度106个/mL,农杆菌OD600值0.6,乙酰丁香酮浓度200μmol/mL,pH 5.8,25℃,共培养4 d...     

农杆菌介导的芒果胶孢炭疽菌遗传转化及致病性缺陷突变体的筛选

构建含潮霉素抗性基因和GFP基因的双元载体pCAMBgfp,以其为转化载体用农杆菌EHA105介导的方法对芒果胶孢炭疽菌Cg-8菌株进行遗传转化,以潮霉素抗性和GFP荧光来筛选阳性转化子。然后,通过离体接种芒果叶片和果实观察病斑大小筛选致病性缺陷转化子。结果获得500个阳性转化子,转化效率为平均106个孢子可获得400个左右同时具有潮霉素抗性和GFP荧光的阳性转化子,且其经多次在无潮霉素的培养基上传代后能得到稳定遗传。随机挑选其中13个突变体进行PCR检测,均可扩增出潮霉素基因目的条带,致病性分析从中筛选得到8个致病性减弱或缺失突变体。     

水稻立枯丝核菌G蛋白β亚基基因的克隆、表达及序列分析

根据Thanatephorus cucumeris G蛋白β亚基序列（AY884129）设计引物,对水稻立枯丝核菌AG 1IA的 G蛋白β亚基基因进行了克隆。PCR结果得到1条约为1.9 kb的扩增片段,包含1个约1.7 kb的完整开放阅读框,编码366个氨基酸。同源性检索发现该序列与大量Ｇ蛋白β亚基基因明显同源,一致性介于57.34%~88.14%。根据其推导cDNA序列设计引物进行RT PCR分析,发现该基因在对数生长期表达量最高,提示水稻立枯丝核菌AG 1IA G蛋白β亚基基因可能具有时空表达特性。     

农杆菌介导的稻瘟病菌转化及致病缺陷突变体筛选

在构建了两个含有潮霉素B磷酸转移酶基因双元载体的基础上,成功地实现了农杆菌介导的稻瘟病菌转化,转化效率达每1×106个分生孢子>300个转化子,并得到了4000多个转化子。通过继代培养和PCR检测,证明插入到稻瘟病菌基因组中的潮霉素抗性基因可稳定遗传。Southern杂交分析表明,大约有2/3转化子的T DNA插入是单拷贝的。用大麦叶片离体接种的方法快速测定部分稻瘟病菌转化子的致病性,发现一个致病缺陷突变体：A1 412。该突变体不能侵入水稻叶片及擦伤的大麦或水稻叶片,说明A1 412突变体在寄主组织中扩展进程被阻断。进一步的表型分析发现A1 412突变体的产孢量显著下降,仅为野生菌株的7%,在疏水表面不能形成附着胞,部分分生孢子萌发也略有延迟。Southern 杂交显示A1 412基因组中T DNA插入是单拷贝的。上述结果表明,A1 412突变体表型的改变可能是由于T DNA插入而使某一具有重要生物学功能的基因失活所致。     

根癌农杆菌介导柱花草炭疽菌遗传转化体系的优化

选取含有氯嘧磺隆抗性标记的ILV1基因和报告基因GFP二元载体的农杆菌AGL-1,采用单因子和双因子方法研究根癌农杆菌AGL-1介导柱花草炭疽菌CH008转化过程中各主要因素对转化率的影响,优化构建ATMT转化柱花草胶孢炭疽菌体系条件,使转化效率达到300 ～400个转化子/106个炭疽孢子,即根癌农杆菌AGL-1浓度OD600=0.8,炭疽菌分生孢子浓度为1x106个/mL,AS浓度为100 μmol/L,诱导时间为6h,共培养温度和时间分别为25 ℃和4d.经PCR检测都有GFP片段,并能够表达绿色荧光蛋白.这为进一步开展炭疽菌对柱花草的致病机理研究提供了依据,优化转化体系有利于后续突变体库的扩大、筛选突变体和致病功能基因的研究.     

油菜黑胫病菌T-DNA插入诱变因素优化及突变体筛选

油菜黑胫病是由Leptosphaeria biglobosa引起的一种真菌病害。这种病害在我国油菜产区广泛发生,并造成一定的经济损失。为通过获取突变体来研究L. biglobosa的生态适应性机制及致病机制,本文优化了影响农杆菌介导转化（ATMT）油菜黑胫病菌L. biglobosa菌株Lb731的因素,评估转化子质量,并筛选相关突变体。结果明确了农杆菌介导转化菌株Lb731的最佳因素：潮霉素B浓度为50 μg/mL,转化受体（分生孢子）培养时间为15 d (20℃),浓度为2 × 10^7~8孢子/mL,农杆菌-受体共培养温度为25℃,共培养时间为72 h。在最适条件下的转化效率达到80个转化子/百万分生孢子。T-DNA插入基因组的频率为100%,单拷贝插入频率为72.7%,转化子抗潮霉素性状能稳定遗传。从2136个转化子中获得了11个菌丝生长减缓突变体,7个色素产生缺陷突变体和14个分生孢子产生缺陷突变体,并从这些突变体中鉴定出7个致病力丧失突变体。采用hiTAIL-PCR技术,从3个突变体中获得了T-DNA插入位点侧翼序列。上述结果为深入研究L. biglobosa的生态适应性机制及致病机制提供了材料和线索。     

橡胶炭疽病菌致病相关突变体的筛选及表型分析

通过对实验室已获得的1 685个橡胶炭疽病菌T-DNA插入突变体的生物学及致病性测定,从中筛选出15个致病力明显减弱的突变体,并进行PCR检测。结果表明,该基因组中均含潮霉素磷酸转移酶基因片段。在PDA培养基上,其中2个突变体菌落颜色异常,6个生长速率下降,8个产孢量显著降低,2个突变体分生孢子形态异常,2个附着胞形态异常,3个不能形成附着胞,在洋葱表皮上,侵染钉形成率显著降低。     

橡胶树尖孢炭疽菌绿色荧光蛋白（GFP）标记转化株的获得

建立了根癌农杆菌介导转化橡胶树尖孢炭疽菌(Collectotrichum acutatum)获得T-DNA插入突变体的体系:在尖孢炭疽菌孢子浓度10~6个/mL、农杆菌浓度OD_(600)=0.15～0.2后在200μmol/mL的乙酰丁香酮(AS)诱导6 h,共培养48 h.转化效率可达150～300个/10~6个分生孢子.获得的转化子通过PCR检测绿色荧光蛋白基因、Southern 杂交验证、分生孢子的荧光观察,结果表明:被测转化子基因组中确实整合了目的片段,成功获得了橡胶树尖孢炭疽菌菌株CHY-1绿色荧光蛋白标记转化子.     

可去除选择标记的转CpTI基因抗虫玉米研究

将组成型表达的玉米泛素启动子与豇豆胰蛋白酶抑制剂基因CpTI连接,插入根癌农杆菌双T-DNA质粒,构建一个T-DNA结构域含有抗潮霉素选择标记基因hyg;另一个T-DNA结构域含有抗虫基因的双T-DNA单子叶植物表达载体,用以转化农杆菌菌株,再通过共培养转化玉米胚性愈伤组织。通过潮霉素培养基抗性筛选,用特异PCR扩增和Southern杂交检测,从分化再生的T0代植株中,鉴定出7个转化CpTI基因的阳性植株。目前,正结合进行田间分离纯合和DNA分子鉴定,培育去除选择标记基因的转基因抗虫玉米自交系。     

农杆菌介导甘蔗基因转化技术的优化

以甘蔗（Saccharum officinarum L.）品种ROC10为转化受体材料,以GFP基因为报告基因,通过对农杆菌介导遗传转化的一系列条件,包括农杆菌菌株及侵染菌液浓度、侵染时间、受体愈伤组织龄期、AS活化时间及使用浓度、共培养时间等进行优化,同时进行了便于vir基因活化和T-DNA转移的条件组合,如附加果糖和葡萄糖、酸性环境感染,低温共培养等,从而建立了一个可行、有效而又简便的农杆菌介导的甘蔗遗传转化体系。结果表明:农杆菌菌株EHA105优于LBA4404,其适宜侵染浓度D600为1.3752;适宜受体愈伤组织龄期为25℃暗培养25d;适宜侵染时间为30min;适宜AS活化时间为2h,使用浓度为200μmol·L-1;适宜共培养时间为4d。获得2株有荧光信号的植株,对这2株植株进行斑点Southern杂交检测,均有阳性反应,证明GFP基因已整合到甘蔗基因组中并得到了有效表达,表明该转化体系确实是有效的。      

一个水稻类病变黄叶突变体的鉴定和精细定位

从15000多个水稻转基因株系中发现了一个类病变黄叶突变体.该突变体最显著的特征为叶片由下而上依次黄化,同时出现类似病原体感染的病斑.根据突变体表型,将该突变体命名为syll(spotted and yellow leayes 1).遗传学分析表明,该突变性状受1对隐性核基因控制.PCR检测和潮霉索抗性分析显示.该突变...     

Three Genes Related to Trehalose Metabolism Affect Sclerotial Development of Rhizoctonia solani AG-1 IA,Causal Agent of Rice Sheath Blight

Trehalose metabolism is related to the sclerotial development of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight (RSB). Here, we further elucidated the functions of three genes Rstre, Rstps1 and Rstpp that encode three key enzymes trehalase (TRE), alpha, alpha-trehalose- phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPP) in the sclerotial development of R. solani AG-1 IA. Due to the lack of a stable genetic transformation system for R. solani, the heterologous expression of these three genes in Pichia pastoris GS115 was performed. The results showed that reactive oxygen species (ROS) contents and enzyme activities in R. solani decreased significantly in the treatments of the fermentation broths of Rstps1 and Rstpp transformants, and that in the treatment of the fermentation broth of Rstre transformant visibly increased. Furthermore, the fermentation broths of the transformants of all the three genes were added to potato dextrose agar (PDA) medium for the cultivation of R. solani, as a result, the dry weight of sclerotia in each PDA plate containing the fermentation broths of Rstps1 and Rstpp transformants significantly increased compared with the control, and that of Rstre transformant obviously decreased. Finally, 178 proteins were found to interact with RSTPS1, and 16 of them were associated with ROS. Taken together, the findings suggest that all these three genes related to trehalose metabolism play important roles in the sclerotial development of R. solani AG-1 IA, and can be used as new targets for the development of novel high-efficiency fungicides for the controlling of RSB.     

转基因橡胶树中载体骨架序列的初步研究

以橡胶树转基因材料为研究对象,首次基于载体左右边界序列设计的特异引物进行载体骨架序列的初步PCR检测,结果表明,15个转基因阳性株系中有5个和2个株系分别含有左侧和右侧的边界序列,占33.3%和13.3%,结合这些株系PCR产物的序列分析结果进一步证实除T-DNA区以外的载体骨架序列确实整合到这些株系的植物基因组中。同时对载体骨架序列存在的弊端及可能的解决途径做了相应的分析。     

Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 μm, He pressure 1100 and 1350 psi with a target distance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5-7 mg L⁻¹) of hygromycin. The integration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T₀ transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function.     

根癌农杆菌介导遗传转化稻曲病菌

利用根癌农杆菌介导转化系统,通过潮霉素抗性筛选转化子,对稻曲病菌分生孢子进行了转化。农杆菌经乙酰丁香酮预先诱导,转化效率大约为390~450个转化子/ 106个孢子。PCR检测绿色荧光蛋白基因和潮霉素基因表达盒,结果显示被测转化子基因组中均成功整合了目的基因片段。同时,在488 nm下这些转化子都具有荧光。表明利用根癌农杆菌可以成功转化稻曲病菌。     

Effect of inoculum density and quantitative PCR-based detection of Rhizoctonia solani AG-2-1 and Fusarium avenaceum on canola

Canola seedling blight, caused by Rhizoctonia solani, and Fusarium spp., can result in large yield losses to canola (Brassica napus) at high inoculum pressure. The effect of inoculum density was studied by mixing different amounts of R. solani AG-2-1 and Fusarium avenaceum into a sterilized natural soil and soil-less mix (2:1, v:v) separately, and recording seedling emergence, damping-off and seedling height within ten days after seeding; root rot severity at 12 days after seeding and seed yield at harvest on canola cultivars ‘45H29’ and ‘73-77RR’. Root rot severity increased and emergence, plant height and seed yield decreased with increased inoculum density of both R. solani and F. avenaceum. For quantification of R. solani AG-2-1, a primer and TaqMan probe set (Rs21F/Rs21R/Rs21P) was designed based on the nuclear ribosomal internal transcribed spacer (ITS) region of R. solani AG-2-1. From a conventional PCR amplification, an 88-bp product was amplified from all isolates classified as AG-2-1 with the primers Rs21F and Rs21R. No product was amplified with DNA from isolates belonging to other anastomosis groups of R. solani, other pathogens or the host plant. By using quantitative PCR, DNA amounts as low as 100 fg of R. solani AG-2-1 were detected. The quantity of DNA from soil samples with different inoculum densities estimated using qPCR was highly correlated to the number of colony-forming units (cfu) obtained from the same soil samples for both R. solani AG-2-1 and F. avenaceum.