Cloning and Analysis of 5' Flanking Region of Sporamin A Gene from Sweet Potato

[Objective] The aim of the study is to clone the 5’ flanking region of Sporamin A gene from sweet potato. [Method] The sweet potato "Xushu 18" was used to amplify the 5’ flanking region of Sporamin A gene using specifically designed primers and then target fragment was analyzed by PLACE and PlantCare online. [Result] Besides the conserved elements including TATA-box and CAAT-box,the cis-acting elements including sucrose responsive element CMSRE1,SP8 acting site and some other regulatory sequence such as MYB binding site were also found in Spo A promoter sequence. The results suggest that the promoter has sucrose responsive function.[Conclusion] The study provided reference to reveal the regulation law of Spo A,and to develop high-level expression vectors promoted by Spo A promoter.     

ISSR-Based Molecular Characterization of an Elite Germplasm Collection of Sweet Potato （Ipomoea batatas L.） in China

To determine the genetic diversity and population structure of sweet potato accessions cultivated in China, and to establish the genetic relationships among their germplasm types, a representative collection of 240 accessions was analyzed using inter-simple sequence repeat （ISSR） markers. The mean genetic similarity coefifcient, Nei’s gene diversity, and shared allele distance of tested sweet potato accessions were 0.7302, 0.3167 and 0.2698, respectively. The 240 accessions could be divided into six subgroups and ifve subpopulations based on neighbor-joining （NJ） clustering and STRUCTURE results, and obvious genetic relationships among the tested sweet potato accessions were identiifed. The marker-based NJ clustering and population structure showed no distinct assignment pattern corresponding to lfesh color or geographical ecotype of the tested sweet potato germplasm. Analysis of molecular variance （AMOVA） revealed small but signiifcant difference between white and orange-lfeshed sweet potato accessions. Small but signiifcant difference were also observed among sweet potato accessions from the Southern summer-autumn sweet potato region, the Yellow River Basin spring and summer sweet potato region and the Yangtze River Basin summer sweet potato region. This study demonstrates that genetic diversity in the tested sweet potato germplasm collection in China is lower than that in some reported sweet potato germplasm collections from other regions. Pedigree investigations suggest that more diverse Chinese sweet potato varieties should be formed by broadening the selection scope of breeding parents and incorporating the introduced varieties into future breeding programs.     

植物HSP70反义基因工程雄性不育表达载体的构建及鉴定(英文)

[Objective]In order to study the relation between the HSP70 gene and male sterility of plant further.[Method]s]Anther specific expression promoter Osg6B of rice was coloned by PCR then connected with HSP70 antisense fragment to construct HSP70 antisense expression vector.The expression vector was identified by PCR experiment and enzyme digestion.[Result]The sequence of coloned Osg6B promoter had 97% homology to the published sequence,and the cis-regulatory element in promoter area was integrated.HSP70 antisense expression vector driven by the promoter Osg6B was confired by colony PCR and enzyme digestion.[Conclusion]The construction of expression vector would lay solid foundation for utilization of genetic engineering male sterility of plant.     

Construction and Identification of HSP70 Antisense RNA Expression Vector for Genetic Engineering Male Sterility in Plant

[Objective]In order to study the relation between the HSP70 gene and male sterility of plant further.[Method]s]Anther specific expression promoter Osg6B of rice was coloned by PCR then connected with HSP70 antisense fragment to construct HSP70 antisense expression vector.The expression vector was identified by PCR experiment and enzyme digestion.[Result]The sequence of coloned Osg6B promoter had 97% homology to the published sequence,and the cis-regulatory element in promoter area was integrated.HSP70 antisense expression vector driven by the promoter Osg6B was confired by colony PCR and enzyme digestion.[Conclusion]The construction of expression vector would lay solid foundation for utilization of genetic engineering male sterility of plant.     

马MxA基因第12外显子多态性检测(英文)

[Objective] The study aimed to establish a fast and accurate method to detect the polymorphism of the 12th exon of equine MxA gene. [Method] The 12th exon of MxA gene was amplified by mismatch PCR and the products were analyzed by restriction fragment length polymorphism (RFLP) to determine the point mutation at the 1 790 nt of MxA cDNA. The sequence of the PCR products was also analyzed. [Result] There were three genotypes (AA, AB and BB) in the 12th exon of equine MxA gene; the 2 081 nt of MxA cDNA mutated from G to C, correspondingly changing the 562th amino acid of the coding region of MxA protein from tryptophan to cysteine; the specific sequence of the PCR products amplified by mismatch PCR-RFLP was consistent with the analysis results of RFLP. [Conclusion] The mismatch PCR-RFLP was an easy method with accurate results to detect the polymorphism of the 12th exon of equine MxA gene.     

Isolation and Characterization of Ell Gene Promoter from Tomato

A fragment of 2000 bp upstream sequence of Ell clone was amplified from genomic DNA of the tomato cultivar Zhongshu- 5. Sequence analysis showed that the upstream contains the regulatory elements： TATA box （-29 - -22）, CAAT box （-193 - -189）, wound, and drought response elements. Expression vectors of Ell promoter gus fusion were constructed with the promoters of 1 200 and 2 000 bp regions, respectively. Transgenic tomato plants were obtained through Agrobacteriummediated transformation. Histochemical analysis of GUS activity in various tissues showed that the two promoters were able to direct fruit-specific gene expression. The expression driven by promoter of 2 000 bp upstream fragment could increase GUS activity with the maturation of tomato fruits. The promoter of -1 200 bp fragment could direct gus gene expression in fruits with the inductions of drought and wounding. The regulatory region for fruit-specificity was probably located in the region of 1 200 bp of 5′-flanking sequence and some positive regulatory elements or enhancers may exist in the region from -1 200 to -2000 bp.     

Research on the Construction of Bacterial Artificial Chromosome Vector DNA and the Potentials in Application

[Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119-Bluelox BAC.[Method] With selecting a proper single restriction site,sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector.[Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection.[Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector,besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.     

一种用高拷贝质粒载体制备BAC载体基本功能基因的新方法(英文)

[Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119-Bluelox BAC.[Method] With selecting a proper single restriction site,sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector.[Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection.[Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector,besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.     

Cloning and Analysis of 5' Flanking Region of Sporamin A Gene from Sweet Potato

[Objective] The aim of the study is to done the 5' flanking region of Sporamin A gene from sweet potato. [Method] The sweet potato "Xushu 18" was used to amplify the 5' flanking region of Sporamin A gene using specifically designed primers and then target fragment was analyzed by PLACE and PlantCare online. [Result] Besides the conserved elements including TATA-box and CAAT-box, the cis-acting elements including sucrose re- sponsive element CMSREI, SP8 acting site and some other regulatory sequence such as MTB binding site were also found in Spo A promoter sequence. The results suggest that the promoter has sucrose responsive function. [Conclusion] The study provided reference to reveal the regulation law of Spo A, and to develop high-level expression vectors promoted by Spo A promoter.     

甘薯贮藏蛋白A基因5''端序列的克隆与分析

甘薯贮藏蛋白A（Sporamin A，spo A）是甘薯块根的主要贮藏蛋白质，占块根可溶性蛋白质总量的60％～80％，位于块根液泡的泡囊中。而在其地上部分的茎中只是微量存在，在叶和叶柄中几乎没有。Ohta等将Spo A基因5’上游的1kb区域与GUS融合，通过农杆菌介导转到烟草中，成功实现了GUS的高活性表达，同时通过SpoA启动子序列截短试验还证明获得GUS基因表达活性的最小长度为305bp。     

甘薯贮藏蛋白A基因5′端序列的克隆与分析

[目的]为开展Spo A基因表达调控规律的研究及利用SopA启动子片段构建甘薯高效表达载体提供理论依据。[方法]应用PCR技术从徐薯18中克隆出甘薯贮藏蛋白A基因5′端1条长度为348 bp的DNA片段,然后应用PLACE、PlantCARE在线启动子预测工具进行分析。[结果]Spo A启动子序列除含有TATA-box、CAAT-box等启动子的保守元件外,还有蔗糖诱导响应元件CMSRE1S、P8作用位点等重要的顺式作用元件,以及一些其他调控序列如MYB结合位点。从序列分析结果可以推测该基因的启动子具有蔗糖创伤诱导响应的功能。[结论]该研究分析和推测了Spo A启动子序列中的作用调控元件,为今后利用该启动子构建甘薯高效表达载体奠定了基础。     

牛SOCS5基因5′调控区多态性研究

【目的】筛选SOCS5基因启动子区多态位点（SNP）,并研究其对启动子功能元件的影响。【方法】选择贵州地方优良品种务川黑牛和中国荷斯坦奶牛两种生长性能差异明显的品种构建DNA池。直接测序后用DNASTAR软件进行序列拼接和校正,BLAST分析SOCS5基因多态性,然后用生物信息学软件预测序列核心启动子区和CpG岛,分析SNP位点对转录因子结合位点影响。【结果】牛SOC5基因5′调控区和第1外显子区存在3个SNP位点,分别为：C-577T、T-43C和C＋61T,其中C＋61T与SNP数据库中的rs110977810信息相符,C-577T和T-43C为新发现SNP位点。生物信息学软件预测得到SOCS5基因核心启动子区和CpG岛,SNP位点导致附近大量转录因子结合位点消失和新位点产生;SNP位点对转录因子结合位点有显著影响,但对核心启动子范围和起始位点无明显影响,不在甲基化水平上影响SOCS基因表达水平。【结论】牛SOCS5基因5′调控区存在3个对启动子功能元件有较大影响的SNP位点。     

猪生长激素启动子的克隆及功能分析(英文)

[目的]克隆猪生长激素启动子,确定其启动子核心序列和主要的顺式作用元件。[方法]根据NCBI上公布的序列设计引物,PCR扩增了猪生长激素5’端-1821~+61bp的序列,并通过移步缺失的方法,获得9段长短不一的启动子序列,将其分别构建到双荧光素酶表达载体pGL3-basic上。通过重组质粒瞬时转染大鼠垂体瘤细胞(GH3)、猪髋动脉血管内皮细胞(PIEC)和猪肾细胞(PK15)和转染后细胞荧光素酶活性的测定,检测这些5’末端缺失质粒在垂体及非垂体细胞中的相对转录活性。[结果]成功扩增了猪GH基因5’上游启动区1882bp的片段并构建了9个pGL3-mGHpromoter报告基因载体;双荧光素酶报告基因检测系统证实插入报告基因载体中的启动子具有非常强的细胞特异性。[结论]猪生长激素特异性在垂体细胞中表达,其最小启动子位于-110bp以内,启动子区-218~-110bp和-429~-218bp间存在正向调控元件。     

贵州矮马与伊犁马生长激素基因5’侧翼区的结构特征

为了摸索贵州矮马生长发育的调控机制,探明贵州矮马与伊犁马生长激素基因5’侧翼区的结构特征,以4～5龄的贵州矮马和7～8龄的新疆伊犁马为研究对象,采用直接测序法从两种马基因组中克隆了生长激素(GH)基因5’侧翼区的DNA序列,并进行生物信息学分析。结果表明:1)从基因组中克隆的711bp片段为GH基因5’侧翼区序列;2)两个马品种的GH基因5’侧翼区中都存在TATA box、CAATbox、GC box和Octamer位点等真核生物启动子结构;3)从伊犁马GH基因5’侧翼区中检测到1个位于30bp处的突变位点发生了G→C颠换,由此新增了1个转录因子upstream stimulatory factor(USF)结合位点;4)从贵州矮马和伊犁马GH基因的5’侧翼区序列中发现了MZF1、CdxA、Nkx-2、AML-1a和c-Myc等19种共58个转录因子的结合位点。结论:克隆的711bp基因序列中存在大量的转录调控元件。     

甘蔗ShSUT4基因及其5'侧翼序列的克隆与序列分析

以甘蔗品种FN28为材料,首次克隆甘蔗ShSUT4基因。测序结果表明,该基因约为4.8 kb,包含5个外显子和4个内含子,并包括完整的开放阅读框。该基因5'侧翼序列长度约为1.8 kb。采用PLACE、PlantCARE在线启动子预测工具分析表明,该序列含有启动子的特定结构,如TATA-box、CAAT-box等,还含有一些顺式作用元件如光响应元件、MYB结合位点、ABRE响应元件等,表明甘蔗ShSUT4基因的表达可能受光照、MYB和ABRE等的调控。     

猪生长激素启动子的克隆及功能分析

[目的]克隆猪生长激素启动子,确定其启动子核心序列和主要的顺式作用元件。[方法]根据NCBI上公布的序列设计引物,PCR扩增了猪生长激素5’端-1 821～+61 bp的序列,并通过移步缺失的方法,获得9段长短不一的启动子序列,将其分别构建到双荧光素酶表达载体pGL3-basic上。通过重组质粒瞬时转染大鼠垂体瘤细胞(GH3)、猪髋动脉血管内皮细胞(PIEC)和猪肾细胞(PK15)和转染后细胞荧光素酶活性的测定,检测这些5’末端缺失质粒在垂体及非垂体细胞中的相对转录活性。[结果]成功扩增了猪GH基因5’上游启动区1 882 bp的片段,并构建了9个pGL3-mGH promoter报告基因载体;双荧光素酶报告基因检测系统证实插入报告基因载体中的启动子具有非常强的细胞特异性。[结论]猪生长激素特异性在垂体细胞中表达,其最小启动子位于-110 bp以内,启动子区-218～-110 bp和-429～-218 bp间存在正向调控元件。     

甘蔗乙烯受体Sc-ERS启动子的克隆与序列分析

【目的】克隆甘蔗乙烯受体Sc-ERS基因启动子序列,分析Sc-ERS基因启动子序列的作用元件。【方法】采用基于热不对称交错式PCR原理的染色体步移技术,从甘蔗ROC22基因组DNA中克隆Sc-ERS基因启动子序列,利用启动子预测软件PlantCARE和PLACE在线工具对Se-ERS启动子序列的作用元件进行预测。【结果】克隆获得sc．ERS启动子序列1410bp,该序列与玉米ERS25、ERS14核苷酸同源性分别为82％和80％。PlantCARE在线分析结果表明,该序列具有启动子基本作用元件TATA．BOX和CAAT—BOX,还含有参与光响应元件、干旱诱导MYB结合位点、茉莉酸甲酯响应元件、水杨酸响应元件、胚乳表达顺式调控元件、热胁迫响应元件等。【结论】从甘蔗基因组DNA中克隆获得乙烯受体Sc-ERS基因上游1410bp的启动子序列,该序列含有多个特异性调控元件,推测sc．ERS;~因的表达可能受生理周期、激素、干旱、光照等因素调控。     

凝固型紫薯酸奶工艺研究

[目的]优化得出凝固型紫薯酸奶的最佳工艺参数,[方法]以紫薯,纯牛乳为主要原料,采用保加利亚乳杆菌和嗜热链球菌混合菌种进行发酵,制备凝固型紫薯酸奶。在单因素的基础上,通过响应面法优化紫薯添加量、蔗糖添加量、接种量和发酵温度4个因素的最佳工艺参数。[结果]以感官评价为指标,紫薯经蒸制,干燥后粉碎并过100目筛以粉状添加时,产品品质较好。紫薯酸奶的最优发酵工艺为：在紫薯添加量为2．2％,蔗糖添加量为6．7％,接种量为10％,发酵温度为41℃时,发酵8h时所得产品色泽均匀,组织细腻,味道酸甜可口,风味纯正,具有浓郁的紫薯香味和酸乳风味。[结论]研究可为市场开发凝固型紫薯酸乳产品提供理论依据,增加功能性酸奶种类,丰富酸奶市场。