粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

利用大肠杆菌克隆在原核生物中有活性的油菜基因启动子

利用本室构建的基因启动子探针型载体ｐＳＵＰＶ４直接在大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ（Ｍｉｇｕｌａ）ＣａｓｔｅｌａｎｉｅｔＣｈａｌｍｅｒｓ）中分离油菜（ＢｒａｓｉｃａｎａｐｕｓＬ．）基因启动子片段,获得卡那霉素抗性重组子３３个。对重组子ｐＲＰ１０做了进一步鉴定：Ｓｏｕｔｈｅｒｎ杂交表明ＲＰ１０片段来自油菜基因组,并与基因组中的另一些序列具有同源性;ＲＰ１０片段５′端区域的缺失可使卡那霉素抗性水平从１００ｍｇ／Ｌ降至２５ｍｇ／Ｌ,说明缺失的序列可能影响基因转录效率;序列分析发现ＲＰ１０片段内存在几个真核基因启动子的保守序列;用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ（ＳｍｉｔｈｅｔＴｏｗｎｓｅｎｄ）Ｃｏｎｎ）转化法将ＲＰ１０ＧＵＳ基因转入油菜,组织化学分析显示ＲＰ１０在愈伤组织中可以启动融合的ＧＵＳ基因表达     

HcNPV半胱氨酸蛋白酶,几丁质酶基因失活分析

将含有美国白蛾核型多角体病毒（Ｈｙｐｈａｎｔｒｉａ ｃｕｍｅａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＨｃＮＰＶ）半胱氨酸蛋白酶基因（ＣＰ）的自然和几丁质酶基因（ＣｈｉＡ）的片段克隆进ＰＣＲⅡ,构建了转移载体ｐＨｃＣＶｄｅｌ;将含有ＨｃＮＰＶ多角体蛋白全基因（ｐｏｌｈ）序列的片段插入到ｐＨｃＣＶｄｅｌ的ＥｃｏＲＩ位点,得到重组转移载体ｐＨｃＣＶｐｏｌｈ。通过重组转移载体与含有家蚕促     

鸡减蛋综合征病毒(EDSV)基因组右末端结构的分析

从中国发病鸡中分离的鸡减蛋综合征病毒（ＥｇｇＤｒｏｐＳｙｎｄｒｏｍＶｉｒｕｓ,ＥＤＳＶ）弱毒株（ＡＡ－２）,其基因组全长约为３３ｋｂ。用限制性内切酶ＨｉｎｄⅢ水解ＥＤＳＶ全基因组,构建了以ｐＢｌｕｅｓｃｒｉｐｔⅡ（ＫＳ＋）为载体的右末端片段的克隆（约４．２ｋｂ）,对其进行了序列测定和结构分析。该片段全长４１８３个碱基对（ｂｐ）,位于基因组右末端８７．３ｍ．ｕ．－１００ｍ．ｕ．。结果显示,该片段与哺乳动物腺病毒右末端Ｅ４区结构不同,与禽Ⅰ型腺病毒代表株ＣＥＬＯ右末端片段亦无同源性。本文为深入了解ＥＤＳＶ基因组结构特点,ＥＤＳＶ与其他腺病毒基因结构与功能的进化关系和ＥＤＳＶ载体的构建奠定了分子生物学基础     

粘虫核型多角体病毒多角体蛋白基因结构和序列的研究

测定了粘虫核型多角体病毒（Ｌｅｕｃａｎｉａｓｅｐａｒａｔａｎｕｃｌｅａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ,ＬｓＮＰＶ）两个ＥｃｏＲＶ片段的共１２０１ｂｐ序列,发现了一个７１４ｂｐ的开放阅读框（ＯＲＦ）,根据其与多种ＮＰＶ多角体蛋白基因（ｏｃｕ）同源性的比较和５＇端典型的１４ｂｐ保守序列,说明此ＯＲＦ即为ＬｓＮＰＶ的ｏｃｕ基因。根据核苷酸序列预测的多角体蛋白有２４６个氨基酸,其中酸性和碱性氨基酸数相等,疏水氨基酸含量很高,氨基酸组成中以谷氨酸（Ｇｌｕ）含量最高,谷氨酰胺和半胱氨酸含量最低。编码区碱基同源性与ＭｂＮＰＶ最高,为９７．０％;氨基酸同源性与ＭｂＮＰＶ最高,为９７．５％。密码子偏爱选用以第三个碱基为嘧啶的密码子。二级结构分析表明,α－螺旋（Ｈ）和β－折叠（Ｓ）相等,β－转角含量是Ｈ和Ｓ含量之和。     

玉米苹果酸脱氢酶基因的分离与结构分析

以一个玉米（ＺｅａｍａｙｓＬ．）杂种一代超亲表达的ｃＤＮＡ片段为探针,从玉米幼苗期ｃＤＮＡ文库中筛选到一个全长１２８７ｂｐ的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ编码细胞质苹果酸脱氢酶,推导的氨基酸序列与龙须海棠（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍ　ｃｒｙｓｔａｌｌｉｕｍ　Ｌ．）及拟南芥（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）同一编码基因的氨基酸序列同源性分别为９０％和８４％。这是禾谷类作物中首次克隆的编码细胞质苹果酸脱氢酶的完整基因。     

不同性别表型黄瓜基因组中雌性系特异的ACC合酶基因

利用一对引物（引物１和引物２）分别从雌性系黄瓜（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ Ｌ．）品种“ＣＯＲＯＮＡ”、“ＤＡＬＥＶＥ”和强雌性黄瓜品种“中农五号”、“欧洲八号”的基因组ＤＮＡ中扩增到一长约１０２５ｂｐ的ＡＣＣ合酶基因片段。序列分析表明：该基因片段与Ｔｒｅｂｉｔｓｈ等１９９７年发表的ＡＣＣ合酶基因片段的同源性大于９９％,认为这两个基因片段应该是同一个基因,不同品种来源的该基因的相同性说明了其高     

水稻重复序列RRD3在转基因植物中的启动子功能

来源于水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）的一个８２０ｂｐ多拷贝重复序列ＲＲＤ３,含有植物启动子ＴＡＴＡ－ｂｏｘ、ＣＡＡＴ－ｂｏｘ等特征保守基元。用ＲＲＤ３取代Ｔｉ载体ｐＢ１１２１中的ＣａｍＶ ３５Ｓ启动子,通过植物转化鉴定ＲＲＤ３的启动子功能。组织化学分析表明,根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ） Ｃｏｎｎ）ＬＢＡ４４０４转化后     

Ⅲ型中国株HCVE2/NS1基因与已知分离株的同源性比较

利用逆转录套式ＰＣＲ扩增Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段,将其克隆到ｐｃＤＮＡ３载体上．采用双脱氧链终止法测定插入片段的核苷酸序列．并与已知分离株的相应区域进行同源性比较．首次克隆出Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因（ＨＣ－Ｗ１４）,其核苷酸序列与Ⅲ型日本株ＨＣＶ（ＨＣ－Ｊ６）该区域同源性为８８．３７％,其推定的氨基酸同源性为８９．２９％．而与已知的非Ⅲ型株ＨＣＶ该区域相比,核苷酸及氨基酸的同源性均相对较低．Ⅲ型中国株ＨＣＶ与Ⅱ型中国株ＨＣＶ在Ｅ２／ＮＳ１区域有较大的变异,揭示研制我国的ＨＣＶ疫苗应该考虑这种基因型之间的变异性．     

番茄花叶病毒外壳蛋白基因及3′端非编码区的克隆、序列分析及其表达

根据已报道的番茄花叶病毒Ｌ株系（ＴｏＭＶＬ）序列人工合成引物,经ＲＴＰＣＲ扩增并克隆了我国番茄花叶病毒分离物（ＴｏＭＶＳ１）的外壳蛋白ＣＰ基因及３′端非编码区。序列测定结果表明,所得ｃＤＮＡ共长６８２个核苷酸,其中ＣＰ基因含４８０个核苷酸,编码１５８个氨基酸,３′端非编码区含２０２个核苷酸,其核苷酸序列与ＴｏＭＶＬ株系具有９９．５％的同源率。将该基因片段克隆到ｐＧＥＭＥＸ１载体中,转入Ｅ．ｃｏｌｉ后诱导表达,经Ｗｅｓｔｅｒｎｂｌｏｔ检测证明,该基因已在大肠杆菌中正确表达。这是我国首次报道ＴｏＭＶＣＰ基因序列。     

一种强启动子的分离与功能

A bidirectional promoter of cotton leaf curl virus (CLCuV) was obtained from the total of DNA CLCuV infected tomato leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into the vector. DNA sequences analysis and homology comparison with the promotor of four kinds of isolates recently found indicated that the cloned promoter fragment composed of 436 bp was 99.32% homolog was up to in nucleotides with that of the isolates. Transient expression vectors were constructed by fusing the promoter fragment with gus reporter gene and nopaline terminator in different orientation. These constructs were delivered into the tobacco (Nicotiana tabacum L.) and cotton ( Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. The results indicated that complementary sense promoter was a strong promoter with high activity in leaf mesophyll and vascular tissues, but virion sense promoter was weaker. The experiments suggested that isolated bidirectional promoter, as a novel strong promoter, could be used for dicots and especially cotton genetic transformation.     

棉花曲叶病毒反式作用因子AC2的功能初探

有关非洲木薯花叶病毒（ACMV）、番茄金色花叶病毒（TGMV）的研究表明,双生病毒编码的反式作用因子AC2反式激活病毒链基因启动子的瞬时表达。以棉花曲叶病毒（CLCuV)侵染的烟草叶片组织总的DNA为模板,通过聚合酶反应扩增CLCuV的AC2基因片段并插入克隆载体。将AC2置于CaMV35S启动子下构建了瞬时表达载体。通过基因他法将质粒载体导入烟草（Nicotiana tabacumL.)和棉花（Gossypium hirstumL.)叶片细胞中进行瞬时表达,结果表明,在反式作用因子AC2的激活下,病毒链基因启动子驱动的GUS活性明显增强,然而激活后的病毒链基因启动子的活性仍低于互补链基因方向启动子;其表达方式与互补链基因启动子相似,即在叶肉及叶脉维管组织均有较高的活性。还探讨了AC2在土壤杆菌介导的转基因植物中的表达行为。     

Preliminary Functional Studies on AC2, a Novel Trans-acting Factor from Cotton Leaf Curl Virus

Studies on tomato golden mosaic virus and African cassava mosaic virus suggested that virion sense promoter was trans -activated in transient expression by A C2 encoded by geminivirus. The AC2 gene fragment of cott on leaf curl virus (CLCuV) was obtained from total DNA of CLCuV infected tobacco leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into vector. Transient expres sion vectors were constructed by fusing the AC2 gene fra gment with CaMV 35S prom oter and nopaline terminator. These constructs were delivered into tobacco [ WT(Nicotiana tabacum L.) and cotton ( Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. Results indicated that activity of virion sense promoter was activated by AC2 and increased remarkably. However, the activity of trans-activated virion sense promoter was still lower than that of complementary sense promoter. Expression pattern of transactivated virion sense promoter was similar to that of complementary sense promoter with the high activity in both mesophyll and vascular of leaf vein. In this paper, the expression behavior of AC2 in Agrobacterium -mediated transgenic plants was also discussed.      

Expression Activity of CLCuV Bidirectional Promoter in Agrobacterium tumefaciens

Cotton leaf curl virus (CLCuV) belongs to the subgroup Ⅲ of geminiviruses with single strand DNA genome. Study demonstrated that the bidirectional promoter of CLCuV had activity in Agrobacterium tumefaciens (Smith et Townsend) Conn. This is the first report for the activity of the bidirectional promoter of geminivirus in A. tumefaciens . Results showed that the activity of the complementary sense promoter was stronger than that of virion sense promoter, and was detected 2-fold higher than that of CaMV 35S promoter in A. tumefaciens . Moreover, the promoter 5′ deletion analysis indicated that the mean GUS activity driven by a 287 nucleotides complementary sense promoter fragment (from -287 to the translation initiation site) is 4 times higher than that driven by the whole complementary sense promoter in A. tumefaciens . This result suggested that there might exist negative regulatory elements in this deleted fragment. The function of other cis- elements included in CLCuV complementary sense promoter was also discussed in this paper.     

Deletional analysis of functional regions of complementary sense promoter from cotton leaf curl virus

Complementary sense promoter from cotton leaf curl virus (CLCuV) is a novel plant promoter for genetic engineering that could drive high-level foreign gene expression in plant. To determine the optimal promoter sequence for gene expression, CLCuV promoter was deleted from its 5' end to form promoter fragments with five different lengths, and chimeric gus genes were constructed using the promoterdeletion. These vectors were delivered into Agrobacterium and tobacco (Nicotiana tabacum L cv. Xanthi) plants which were transformed by leaf discs method. GUS activity of transgenic plants was measured. The results showed that GUS activities with the promoter deleted to -287 and -271 from the translation initiation site were respectively about five and three times that of full-length promoter. There exists a c/s-element which is important for the expressing activity in phloem from -271 to -176. Deletion from -176 to -141 resulted in a 20-30-fold reduction in GUS activity in leaves with weak activity in leaves and     

Promoter Function of a Rice Repetitive DNA Sequence RRD3 in Transgenic Plants

A 820 bp rice (Oryza sativa L.) repetitive DNA sequence, the RRD3, was cloned by annealing kinetics. From the sequence analysis, there are several conserved promoter motifs in the sequence such as TATA-box, CAAT-box, etc. In order to detect the promoter function of RRD3, RRD3 was inserted into Ti plasmid pBI121 to replace the CaMV 35S promoter DNA fragment. Both transgenic tobacco (Nicotiana tabacum L.) G28 and rice callus showed the β-glucuronidase (GUS) activity by histochemical assays, the GUS activities of the transgenic tobacco were primarily localized at or around the vascular tissue in leaf and stem. These results indicated that RRD3 can exercise promoter function. The core sequence of promoter of RRD3 will be located.     

菠菜磷酸丙糖转移蛋白在烟草中正义及反义表达及其对碳水化合物转运的影响

以菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）为材料,取幼叶分离ｍＲＮＡ,反转录合成ｃＤＮＡ,以ｃＤＮＡ第一链为模板,通过ＰＣＲ扩增,获得菠菜磷酸丙糖转移蛋白（Ｔｒｉｏｓｅ ｐｈｏｓｐｈａｔｅ ｔｒａｎｓｌｏｃａｔｏｒ,ＴＰＴ）ｃＤＨＡ目的片段。对其进行序列分析,结果表明,分离的目的片段核苷酸序列与文献报道相比同源率为９９．９％,只不１个碱基发生改变。将得到的菠菜ｔｐｔ ｃＤＮＡ与ＣａＭＶ３５     

Cotton Leaf Curl Virus: lonic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus (CLCuV) in two cotton (Gossypium hirsutum L.) cultivars (i.e. CIM-240 and S-12) was investigated. The incidence of disease attack was found to be 100% in the S-12 cultivar and 16% in the CIM-240 cultivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antisera-treated cell sap. The CLCuV impaired the accumulation of different nutrients in both cultivars. A marked decrease in the accumulation of Ca2+ and K+ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na+, N, and P. It was observed that the curling of leaf margins in CLCuV-infected plants was associated with the leaf Ca2+ content; leaf curling was severe in plants with a significant reduction in Ca2+ content.Moreover, leaf K+ content was found to be associated with resistance/susceptibility to CLCuV infection.