农杆菌介导的黄瓜炭疽菌遗传转化

建立并优化了农杆菌介导转化瓜类炭疽菌(Colletotrichum lagenarium)获得T-DNA插入突变体体系,包括农杆菌使用浓度、农杆菌与炭疽菌孢子共培养时间、抗生素配合使用抑制农杆菌污染等.所用的转化载体pBIG2RHPH2-GUS-GFP除了抗生素选择标记外还携带有融合GFP-GUS报告基因.转化106个孢子平均可获得约150～200个潮霉素抗性转化子.PCR检测表明,所有潮霉素抗性转化子菌株都能从其基因组DNA中扩增到转化载体中的GUS基因片段,而且都能产生GFP荧光或GUS染色阳性.转化子菌株经过连续继代培养后保持稳定.随机对36个转化子菌株进行致病性测定,发现1个转化子菌株对黄瓜致病性下降,1个丧失致病性,而大多数表现为野生型致病性.农杆菌介导转化瓜类炭疽菌体系的建立,为进一步构建大库容量的T-DNA插入突变体库、致病性突变体筛选以及致病性相关基因的克隆鉴定奠定了基础.     

稻瘟菌若干T-DNA插入突变体初步分析

为揭示稻瘟菌致病分子机制,创建了该菌T-DNA插入突变体库．部分突变体经表型分析,获得3个生长发育及致病性同时发生变异,5个生长发育正常但致病性变异及2个生长发育变异但致病性正常的突变体；以TAIL-PCR方法获得了这些突变体T-DNA插入位点及其边界序列,并用生物信息方法对被T-DNA标签的基因进行了分析,为进一步研究这些基因的功能提供了重要信息．     

Endoscopically guided nasojejunal tube placement in dogs for short‐term postduodenal feeding

Objective – To evaluate a method for endoscopically guided nasojejunal tube placement allowing short‐term postduodenal feeding and chyme withdrawal in dogs. Design – Pilot study. Setting – University teaching hospital. Animals – Three healthy Beagle dogs with jejunal nipple valve fistulas. Interventions – After the dogs were anesthetized, an 8 Fr, 250‐cm polyvinyl chloride catheter was advanced through a gastroscope into the jejunum. Correct jejunal placement was established using endoscopic visualization and confirmed by fluoroscopy and radiography. The proximal end of the tube was pulled out through 1 nostril and sutured to the skin of the forehead. Thereafter, jejunal feeding was administered for 4 days. Follow‐up examinations included daily confirmation of the tube's position using radiography, physical examination, and blood analyses. Withdrawal of jejunal chyme was performed after jejunal and oral feeding. Measurements and Main Results – Fluoroscopic examination confirmed that endoscopic visualization alone allowed correct jejunal placement. During a 4‐day postduodenal feeding period, repeated radiographic examination revealed stable positioning of the tubes within the jejunum with minor cranial displacement. The tubes were functional throughout the study without causing identifiable problems. Repeated physical examinations and blood analysis showed no abnormalities. We were able to administer the daily caloric requirements as a liquid diet. Jejunal chyme was successfully withdrawn via the tube. Conclusions – Endoscopically guided nasojejunal tube placement was shown to be a minimally invasive, well‐tolerated method for short‐term jejunal feeding in healthy dogs. This technique is a viable option for dogs requiring jejunal feeding but not laparotomy. The feasibility of chyme sampling is another unique application of the procedure.     

T-DNA (Ds) 插入产生的水稻卷叶突变的遗传分析

在筛选和鉴定水稻T-DNA（Ds）插入纯合体的过程中，观察到1个叶片卷曲的突变体．对该突变体进行遗传分析表明，分离群体出现叶片卷曲和叶片正常2种植株类型，其分离比率为3：1，符合1对基因的显性遗传．Basta抗性检测及PCR分子检测证实，该突变体是由单一T-DNA（Ds）插入所引起的，突变性状与T-DNA（Ds）共分离．该突变材料可用于插入座位的基因克隆．     

利用农杆菌转化花椰菜组织的研究

供试的四个花椰菜品系对农杆菌T_(37)反应极为敏感,但各品系反应程度有所不同,肿瘤发生率变动于64%-82%之间。利用含有二元载体的农杆菌LBA_(4404)与无菌幼苗的下胚轴切段共培,获得卡拉霉素抗性植株。DNA分子杂交证明,二元载体上的T—DNA确实整合到植物染色体上,其npt嵌合体基因使转化体具有卡拉霉素抗性。由胭脂碱Ti质粒pTiT_(37)诱导的丛生芽,通过组织培养较易分化出不定根,但这些芽状结构很难发育成正常植株。     

利用二代测序技术对柔嫩艾美耳球虫T-DNA整合位点的分析及鉴定

为建立一种适用于转基因球虫的快速且准确的分析T-DNA整合位点的方法，本研究利用二代测序技术对转基因柔嫩艾美耳球虫EtM2e虫株进行基因组重测序分析，基于嵌合体reads比对分析T-DNA的插入位点，通过T-DNA特有序列以及外源基因与球虫同源序列的测序深度进而分析T-DNA拷贝数，最后利用PCR扩增验证分析位点。结果表明:1)二代测序下机数据过滤后获得数据7.2 Gb，Q20为96.1%，Q30为89.2%，能够比对至柔嫩艾美耳球虫参考基因组的reads数为23 992 361×2，平均测序深度为80×，reads全基因覆盖结果表明整个染色体被覆盖的较为均匀，测序的随机性比较好，可以进行后续分析；2)比对至T-DNA序列的reads数为3 106和3 036，平均测序深度为140×，嵌合体reads序列信息表明T-DNA只存在一个插入位点，位于HG994969.1:2 704 213~2 705 255；3)T载体骨架特有序列卡那抗性基因的平均测序深度为67×，T-DNA特有序列EYFP基因的平均测序深度为85×，而T-DNA与球虫同源序列His4基因的5′调控区序列的平均测序深度为154×，Actin基因的3′调控区序列的平均测序深度为164×，同源序列的平均测序约为特有序列平均测序深度的2倍，这表明T-DNA为单拷贝，与发现一个插入位点的结果相符合；4)经PCR验证分析成功鉴定了该整合位点。转基因艾美耳球虫EtM2e虫株T-DNA整合位点的鉴定为后续转基因球虫鉴定提供了技术平台，也为球虫活载体疫苗的研发提供了一个潜在的靶位点。     

水稻T-DNA插入氮营养缺陷型突变体的初步筛选

通过温室水培试验对根癌农杆菌介导的T-DNA随机插入构建的水稻突变体库的T1代2 173个家系进行筛选.以叶绿素SPAD值为指标,以苗期表观症状为参考指标,筛选到27个氮营养缺失的突变家系.对27个突变家系进行遗传分析、复筛及T2代鉴定,结果表明,编号955家系为氮营养缺陷型突变家系,且T2代能够稳定遗传.该材料通过进一步鉴定可用于分离、鉴定氮营养相关基因.