绿色荧光蛋白基因银耳表达载体构建及表达

以银耳芽孢为材料,由载体pEGFP、pCAMBIA1300经过HindIII和EcoRⅠ双酶切、连接、转化,重组质粒酶切验证,成功构建了由双孢蘑菇gpd启动子调控EGFP基因的银耳真核表达载体,命名pCB-BEGFP。采用电击转化法将携带EGFP基因的银耳表达载体pCB-BEGFP转化到银耳芽孢。提取4个转化子基因组DNA,用EGFP基因特异引物PCR扩增,电泳结果表明有与EGFP基因大小一致的特异带出现;荧光显微镜观察在再生培养基上的转化子可看到很强的绿光;其中一个转化子蛋白SDS-PAGE电泳,结果发现在大约27kD处有一明显的蛋白条带出现,与预期的蛋白分子量相近。以上结果证明EGFP基因转入银耳芽孢中,双孢蘑菇启动子可以调控外源基因的在银耳芽孢中正确表达。     

风信子DFR基因全长cDNA的克隆及序列分析

以蓝色风信子完全露出蓝色的花蕾为材料,利用RT-PCR和RACE技术，获得风信子DFR基因的全长cDNA。该cDNA全长1252 bp ,开放阅读框为1098 bp ,编码366个氨基酸。 Blast搜索结果显示,风信子DFR基因核苷酸序列与其它植物已报道的DFR基因具有66 %～77 % 的相似性,氨基酸序列有62 %～73 %的相似性。聚类分析表明, 最先与风信子聚类合并的是鸢尾，其次与其它单子叶植物聚类，最后与双子叶植物聚类。     

水稻野败型细胞质雄性不育恢复基因Rf3的定位

以珍汕97A/明恢63的F2群体为材料,应用SSR标记对水稻野败型恢复基因Rf3进行定位。该试验从F2分离群体中筛选出119个极端不育单株组成隐性基因定位群体。针对水稻第1染色体短臂Rf3所在染色体的可能区间,应用37个SSR标记检测亲本,从16个多态性标记中挑选出9个检测定位群体。结果表明物理位置连续排列的SSR标记RM10353、RM1195和RM3746各有8个单株与Rf3基因发生了单交换,且重组子数表现为最少,据此可将Rf3定位于这3个标记的两侧标记内。因此最终将Rf3定位在相距679.9 kb的SSR标记RM10338和RM10376之间。     

Mapping of Rice Fertility-Restoring Genes for ID-Type Cytoplasmic Male Sterility in a Restorer Line R68

An F2 population derived from the cross Zhong 9NR68 was used to map the fertility-restoring (Rf) gene for ID-type cytoplasmic male sterility (CMS).Two bulks (a fertile bulk and a sterile bulk) were constructed by pooling equal amount of ten highly fertile lines and ten highly sterile lines,respectively.Four hundred and thirteen pairs of simple sequence repeat (SSR) primers,which evenly distributed on 12 chromosomes of rice,were selected for analyzing polymorphisms between the parents and between the two bulks.The primer RM283 on chromosome 1 and the primers RM5756,RM258,RM6100 and RM171 on chromosome 10 were found to be polymorphic between the parents and between the two bulks.These five SSR markers were linked to fertility-restoring genes.A total of 82 excessive sterile lines were selected from Zhong 9NR68 F2 population to estimate the genetic distance between five SSR markers and fertility-restoring genes respectively.The results indicated that one Rf gene was linked to RM283 located on chromosome 1 at a distance of 6.7 cM,and the other Rfgene was mapped to the long arm of chromosome 10 flanked by RM258 and RM6100 at the distances of 8.0 cM and 2.4 cM,respectively.     

犬瘟热病毒核衣壳蛋白N基因的真核表达及鉴定

根据GenBank发表的犬瘟热病毒N基因全序列,设计合成了1对特异扩增CDVN基因的引物。以山东泰安分离的CDV-FOX-TA株细胞毒中提取病毒RNA来制模板,利用RT-PCR扩增出了1.6kb的N基因,将其克隆到pIREShyg载体上,构建了pIRES-N真核表达载体。然后通过磷酸钙共沉淀法转染CHO-K1细胞,通过潮霉素筛选得到阳性克隆,间接免疫荧光实验（IFA）鉴定N基因在CHO细胞中的表达,并用RT-PCR方法从转录水平证实N基因在CHO-K1细胞中的表达,最终建立了CHO/CDV-N细胞株,为犬瘟热病毒的血清学检测和基因疫苗的研制奠定了基础。     

1BL/1RS小麦的分子和细胞学鉴定及黏类CMS育性恢复区域分布的分析

【目的】揭示黏类小麦细胞质雄性不育系育性恢复基因的区域分布及恢复材料的1BL/1RS情况,为黏类细胞质雄性不育系优良恢复源筛选提供理论依据,以推动黏类小麦雄性不育"三系"强优势组合的选配能不断得到新的材料保障。【方法】以8个黏类不育系和国内外一批小麦品种(系)为试材,结合分子和细胞学技术,进行1BL/1RS易位系鉴定,并利用中国国内法对其育性恢复程度进行分类。【结果】在参试的256份材料中,初步鉴定约20%的小麦品种(系)属于1BL/1RS易位系;育性表现半不育、高可育和全可育的品种(系)分别有86.15%、91.67%和100%为非1BL/1RS易位系,表现全不育和高不育的品种(系)中均有40%左右属于1BL/1RS易位系;恢复能力在50%以上的品种(系)在中国春麦区、长江中下游冬麦区、西南冬麦区和华南冬麦区的比例依次为60%、65.85%、68.42%和71.43%。【结论】黏类不育系优良恢复源大都为非1BL/1RS易位系,主要集中在中国春麦区和南方冬麦区。     

拟南芥种皮色素形成机制研究进展

类黄酮为拟南芥种皮的主要成色物质,其生物合成过程受到一系列基因的影响.这些基因中,一部分为结构基因(TT4、TT5、TT6、TT7、FLS1TT3,LDOX 和BAN)编码一些酶类参与到类黄酮的生物合成;一部分作为调控基因(TT1, TT2, TT8, TTG1, TTG2 和TT16)编码一些酶对生物合成途径起调控作用;另外一些基因（TT12, TT19）编码与色素转运、积累相关的蛋白质。这些基因中的一种或几种发生变异就能影响到类黄酮的生物合成,从而引起拟南芥种皮色泽的变异。在此,就拟南芥种皮的色泽变异及类黄酮生物合成机理作一介绍。     

抗虫转基因植物的鉴定方法研究进展

农作物受害虫侵害严重,给农业生产带来巨大损失,喷施化学杀虫剂使害虫产生抗药性,且污染环境。近年来,转基因技术研究不断深入,抗虫转化研究工作进展迅速。鉴定转基因植物的方法技术有很多种,在此主要阐述了利用标记基因进行鉴定,分子标记,免役技术三大类方法,并对其原理分别进行了简单介绍。     

Isolation and genetic characterization of a fertility-restoring revertant induced from cytoplasmic male sterile rice

Summary A male fertile revertant was isolated from M₁ of a cytoplasmic male sterile indica rice line II-32A, the dry seeds of which were treated with ⁶⁰Co- rays at a dose of 290 Gy. The acquired revertant T24 was morphologically and agronomically similar to II-32B, the maintainer of II-32A. Testcrosses of the revertant with II-32A and Zhenshan 97A showed that the revertant was able to restore the fertility of CMS lines. Genetic analysis of the progenies between T24 and II-32A, Zhenshan 97A XieqingzaoA and DZhenshan 97A, which have different cytoplasms, showed that the fertility restoration of four CMS lines by T24 involved one nuclear gene, indicating that T24 was a result of the mutation of one nuclear gene. The mechanism of the restoration of CMS line by T24 was obviously different from other restorers such as Minghui 63 and 20964, which were shown to behave in two gene mode in fertility restoration. The discovery of the revertant T24 contributes to the understanding of CMS and fertility restoration of CMS in rice. The T24 and its parent II-32A may constitute a pair of near isogenic lines for the restoring gene, which should be valuable materials for molecular genetic analysis of CMS.     

SSR and SCAR markers linked to the fertility-restoring gene for a D2-type cytoplasmic male-sterile line in wheat

‘Yi 4060’ is an elite restorer line of a non‐photoperiod‐sensitive D²‐type cytoplasmic male‐sterile (CMS) line of wheat. Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were employed to map one major fertility‐restoring gene (D²Rf₁) in ‘Yi 4060′. The sterile and fertile DNA pools were established from individuals in BC₆, based on bulked segregant analysis. One RAPD marker E09, linked to D²Rf₁, was converted to a SCAR marker and designated as E09‐SCAR₈₆₅. The genetic distance between E09‐SCAR₈₆₅ and D²Rf₁ is 9.5 cM. Two SSR markers, Xgwm11 and Xgwm18, were also linked to a D²Rf₁ and co‐segregated with E09‐SCAR₈₆₅. The three molecular markers are useful in marker‐assisted breeding of the elite restorer lines for D² ‐type CMS lines in wheat.