番茄果实特异性启动子2A11的基因克隆及功能研究

采用巢式PCR技术从番茄基因组DNA克隆到长度1.3kp的果实特异性2A11启动子基因。序列分析表明,克隆到的基因序列2A11启动子转录起始位点上游的621bp处缺失了已报道的番茄2A11启动子基因（GenBank ID M87659,1993）序列中的“tatattgttaacttcttgttgaattaaagcaat”片段,其同源性为61%,登入GenBank,ID号为DQ453963;构建植物表达载体pCAMBIA2A11,用农杆菌介导侵染番茄果实,GUS基因瞬间表达结果表明,该2A11启动子基因具有驱动GUS基因在番茄果实中特异性表达的功能。研究结果表明成功地获得2A11果实特异性启动子基因,为下一步转基因番茄口服疫苗的研制奠定了一定的基础。     

橡胶树白粉菌内源强启动子WY193的克隆及功能鉴定

橡胶树白粉菌(Oidium heveae Steinm.)是专性寄生的丝状真菌。目前,有关橡胶树白粉菌启动子的研究较为落后。启动子是调控基因表达重要的顺式作用元件,是分子生物学研究的热点。为了开发研究橡胶树白粉菌的内源启动子,为植物基因工程提供新的材料及从分子水平理解橡胶树白粉菌遗传组成与进化。本研究在橡胶树白粉菌HO-73基因组基础上,利用生物信息学在线软件PromoterScan预测得到一个疑似启动子,命名为WY193。采用qRT-PCR技术测定WY193调控GUS基因的表达量,其表达量明显高于阳性对照CaMV 35S(35S)启动子,为35S的8.34倍。WY193均能驱动GUS基因在双子叶植物烟草和火龙果,以及单子叶植物椰子和玉米中进行瞬时表达。因此,本研究得到的橡胶树白粉菌内源启动子WY193,其表达范围较广,效率高,具有较好的应用前景,丰富了橡胶树白粉菌内源启动子研究。     

Analysis of puroindoline a and b sequences from Triticum aestivum cv. `Penawawa' and related diploid taxa

Flanking sequences of the puroindoline a(pinA) and puroindoline b (pinB) genes from Triticum monococcum, T. urartu, Aegilops speltoides and A. tauschii were obtained by inverse PCR. Two accessions from each of these related diploid taxa were sequenced, and the sequences compared to those of the soft hexaploid wheat cultivar `Penawawa'. As expected from the origin of the D-genome in hexaploid wheat, the highest sequence identity was observed for the A. tauschii sequences, whereas the sequences for A.speltoides were the most divergent. The promoter sequences were further analyzed for putative regulatory elements, and the possible significance of several highly conserved sequence motifs is discussed. Western blots of Triton X-114 extracted proteins separated by SDS-PAGE confirmed the accumulation of pinA and pinB gene products in the endosperm of all the germplasm lines studied. The deduced amino acid sequences for the related diploid taxa do not imply any major structural changes as compared to the wild-type T. aestivum puroindolines, and is therefore in agreement with the soft endosperm texture of these species. This revised version was published online in August 2006 with corrections to the Cover Date.     

小麦类发芽素蛋白3启动子序列(登录号:AY864922)

In germinating wheat embryos, gl-OXO accumulation is localized in cell wall. It has been confirmed that this enzyme locally provides H2O2 to catalyze peroxide-mediated cross-linking of cell wall components in terminal cellular differentiation and plays an important role in enabling cells to retain their meristematic and organogenic capacity. Using tail-PCR and DNA sequencing techniques, we isolated Germin-like Protein 3 promoter sequence（l 654 bp）, from common wheat cultivar （yumai 18） genome. No GGGCGGG sequence exiting in promoter implies that germin-like protein 3 is not “house-keeping” protein. The presence of TGTCTC, an auxin response element and localizing at upstream -258, indicates that this promoter is auxin-inducible. The TATA box situates in upstream -27- -32 and 5＇-UTR consists of 95 bp.     

Leaf yield component combining abilities in mulberry (Morus spp.)

A line × tester analysis was carried out in mulberry (Morus spp.) to determine the genetic interaction in the expression of various quantitative characters including leaf yield. Eight clonal varieties were selected, 3 of them were designated as lines (♀) viz. Berhampore-1, China white and MS-5 and 5 of them were called as testers (♂) viz. Mandalaya, Kosen, Assamjati, MS-1 and Kajli. Combining ability studies were conducted on these parents along with their F₁ hybrids for the variables laminar index, growth rate, weight of 100 dry leaves, number of primary branches per plant, plant height, nodal distance, leaf-twig ratio, aerial biomass, moisture content, moisture retention capacity and leaf yield. Broad genetic variability was observed among the genotypes. The ratio of General Combining Ability (GCA) and Specific Combining Ability (SCA) indicated the predominance of non-additive genes in mulberry. While China white (female) and MS-1 (male) were the best general combiners among the parents, Berhampore-1 × Kajli was the best cross for leaf yield. Results suggest that selective crossing followed by proper screening may be the best approach for breeding of high yielding varieties in mulberry. This revised version was published online in July 2006 with corrections to the Cover Date.     

橡胶树延伸因子cDNA及其5′端启动子区域序列的分离与分析

橡胶延伸因子REF(Rubber Elongation Factor)是橡胶生物合成中最重要的酶之一, 是胶乳中主要的蛋白质. 作者利用已发表的REF cDNA序列设计并合成引物. 通过提取胶乳总RNA用RT法合成cDNA后, 通过PCR技术扩增并克隆了REF cDNA序列. 序列测定结果表明, 所克隆的REF cDNA编码区序列全长414个碱基, 编码138个氨基酸, 3′端非编码区     

抛栽密度与促芽肥用量和化学促芽剂对中稻—再生稻的影响研究

此研究是针对再生稻生产中存在发苗不足、成穗率低、肥料利用率不高等因素而设计。试验结果表明,抛栽密度在18～31.5万穴/hm^2的范围内,再生稻粒重变化不大,实际产量基本一致,但再生稻发苗数和有效穗以18～22.5万穴/hm^2最高,分别达20.3～24.3苗/穴和18.9～20.1穗/穴;促芽肥施用过多,会影响头季中稻的产量,随促芽肥的增加,再生芽长也增长,但发苗数、有效穗数、结实率、单穴穗重则有下降趋势。同时,化学促芽剂4PU-30、高产精、腐殖酸、IAA能够增加头季中稻单穗重和促进再生芽的萌发生长,提高穗数和产量。     

植物提取物应用于水产饲料的研究进展

植物提取物因其安全、绿色和高效等特点已成为目前替代抗生素的研究热点。植物提取物含有多种有效成分,具有多种生理活性,应用于不同水产动物效果不同。本文分别从诱食性、促生长、免疫增强、抗氧化应激和抗病原菌五个方面,综述了植物提取物在水产饲料中的最新研究进展,为开发环保型饲料添加剂提供理论参考。     

小麦穗发芽抗性相关Vp1基因启动子的分离及功能验证

成熟期穗发芽严重影响小麦产量和品质。Vp1是调节胚发育, 促进胚成熟和休眠的重要转录因子, 对小麦种子休眠和穗发芽抗性具有重要作用。本研究分离了普通小麦B基因组Vp1基因的启动子, 生物信息学预测结果表明, 其含有9个脱落酸响应元件ABRE、2个DREB和6个MYB干旱响应元件、3个赤霉素响应元件GARE、1个水杨酸响应元件TCA-E、2个茉莉酸甲酯响应元件TGACG-motif、4个SKn-1和1个RYREPE胚乳特异表达元件。采用5′端缺失的方法, 构建了系列含Vp1启动子不同区段融合GUS报告基因的瞬时表达载体和植物表达载体。通过基因枪转化小麦愈伤组织, 瞬时表达结果显示, Vp1启动子在无诱导的情况下不能启动GUS基因表达, 在低温、ABA、GA、PEG和NaCl诱导后可以启动GUS基因表达, 表现诱导表达特性, 且其诱导表达强度随启动子缺失片段长度变短而减弱。利用Gateway方法成功构建了6个启动子各缺失片段类型的植物表达载体, 并通过农杆菌介导转化四倍体小麦Stewart, 获得转基因植株。该启动子可有效启动GUS基因在转基因植株的花药、糊粉层、穗轴及根中表达, 其他组织中没有表达。当启动子片段大于660 bp时, 外源ABA可诱导启动子启动GUS基因在转基因植株茎节中的表达。     

马铃薯损伤诱导型启动子Wun1基因的克隆及其GFP表达活性

通过聚合酶链式反应(PCR),以马铃薯基因组DNA为模板,根据已报道Wun1序列设计了一对特异引物,在优化的PCR反应条件下扩增出了Wun1基因片段,通过序列分析与文献报道的碱基序列有96.86%的同源性,该基因已登录到GenBank(No.AY803296)。以pBIPG(携带GFP基因)的质粒DNA为模板,通过PCR技术亚克隆到了源自水母(Aequorea)大小为756bp的绿色荧光蛋白(GFP)基因,与已知序列同源率为100%。利用GFP基因作为报告基因,构建了用于比较鉴定所克隆启动子活性的pBIG(35S-GFP)和pBIWG(Wun1-GFP)两个植物表达载体,采用基因枪法进行对洋葱表皮细胞的遗传转化,检测Wun1启动子在受体细胞中调控基因表达的活性,结果表明克隆到的Wun1启动子活性强于组成型表达的35S启动子,GFP瞬时表达的分析方法也让我们有效的筛选到用于进行马铃薯抗病育种的调控元件。