大蕉冷诱导差减文库的构建与分析

以低温处理的大蕉幼苗cDNA为检测子,未处理的大蕉幼苗cDNA为驱赶子,利用抑制性差减杂交技术(suppression subtractive hybridization,SSH)构建了大蕉幼苗冷诱导差减cDNA文库,通过点杂交差异筛选cD-NA文库,得到约50个低温下表达增强的候选克隆,对其进行DNA测序和同源性比较,发现获得的ESTs在功能上主要涉及信号传导、表达调控、非生物胁迫防御、蛋白质加工和能量代谢等方面。该SSH文库的构建为克隆大蕉抗寒相关基因奠定了基础。     

褐飞虱细胞色素P450基因cDNA片段的克隆、测序及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱细胞色素P450基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为237bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自烟草天蛾、棉铃虫、埃及伊蚊、家蝇、黑腹果蝇和线虫的CYP6家族的P450的氨基酸序列存在同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,P450基因的表达水平明显升高.以上结果表明,P450基因的表达受抗性水稻的诱导,该基因在褐飞虱对抗性水稻的耐受性和解毒方面可能起着重要作用.     

褐飞虱羧酸酯酶基因cDNA片段的克隆及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱羧酸酯酶基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为396 bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自铜绿蝇、家蝇、沟鼠、黑腹果蝇、线虫和埃及伊蚊的羧酸酯酶的片段存在高度同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,羧酸酯酶基因表达水平明显升高.以上结果表明,羧酸酯酶基因的表达受抗性水稻的诱导,该基因在有毒化学物质解毒及增强褐飞虱对抗性水稻的耐受性方面可能起着重要作用.     

95%烯丙异噻唑原药

烯丙异噻唑;是防治水稻稻瘟病的药剂,采用糖精、烯丙醇氯化亚砜等原料,经氯化醚化反应得到烯丙异噻唑原药总收率为82%以上,含量在95%以上,其工艺可行,设备选型合理,产品属国内领先.     

花椰菜（Brassica oleracea vat．botrytis）抗黑腐病差异表达cDNA片段的克隆及功能的初步研究

采用花椰菜抗黑腐病近等基因系C731（感病系）和C712（抗病系）作为材料,利用cDNA—AFLP技术,研究了花椰菜黑腐病抗性在黑腐病菌侵染和非侵染条件下基因表达的情况．获得了两个阳性克隆M2和M6．Northern杂交和点杂交进一步证明M2是组成型表达的cDNA片段,只是在病菌侵染与非侵染条件下表达丰度不同．而M6是差异表达的cDNA片段．Southern杂交表明M6 cDNA片段在花椰菜基因组中是单拷贝序列．随后的序列分析发现M6cDNA片段与拟南芥1号染色体的BACF19P19中66665～66813bp有84％同源性,该片段编码拟南芥的2A6蛋白的部分序列．推测的氨基酸序列与拟南芥的2A6蛋白部分序列有91％同源性．用H2O2作为外源分子胁迫处理的初步功能分析发现：M6cDNA片段受H2O2诱导,在诱导早期16～24h高度表达,其在叶片中的积累呈逐渐上升趋势．根据序列分析和初步的功能分析的结果推测：M6cDNA片段可能就是编码花椰菜中类2A6蛋白的部分基因片段．是参与花椰菜抗病反应信号途径的相关调控基因片段．     

水稻蔗糖转化酶基因的克隆及其功能的初步探讨

根据抑制消减杂交(suppression subtractive hybridization，SSH)方法分离到在水稻叶片中高表达的蔗糖转化酶基因片段，根据水稻基因组顺序设计引物分离到全长cDNA．测序结果表明该cDNA长度为1937bp，推测编码一个627个氨基酸的中性/碱性蔗糖转化酶．Alignment分析显示该推测蛋白质与已知的多个蔗糖转化酶具有很高的同源性．半定量PCR检测证实它在叶中的表达强于在根、花药和幼穗等组织中的表达，胁迫处理(盐和低温)使其表达上调．     

用差减cDNA文库筛选由热激导致的小鼠睾丸中差异表达的基因

采用抑制性差减杂交（Suppression Subtractive Hybridization, SSH）方法,构建正常和热激小鼠睾丸组织的差减 cDNA 文库,以期 筛选出小鼠生精过程中对热敏感的基因。分别从正常和热激小鼠睾丸组织提取总RNA,反转录成cDNA,以正常小鼠睾丸组织cDNA作为待检组织(tester),以热激小鼠睾丸组织cDNA作为驱动组织(driver), 经过2轮杂交和抑制性PCR扩增,产物与T载体连接,经蓝白斑筛选,提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了2种组织间差异表达基因的差减cDNA文库。用半定量RT-PCR方法,进一步验证 了该文库中差减出的基因基本上为差异表达的基因。对随机挑选其中的932个克隆测序,对有效测序的565个基因序列与GenBank数据库中发布的序列进行同源性比较,大部分片段都可以检索到同源序列。 结果显示,cPGES/p23等13个基因热激后表达量上调;septin2等120个基因热激后表达量下调。其中cPGES/p23为本研究中首次发现的小鼠生精过程中对热敏感的基因。     

花椰菜(Brassica oleracea var.botrytis)抗黑腐病差异表达cDNA片段的克隆及功能的初步研究

采用花椰菜抗黑腐病近等基因系C731(感病系)和C712(抗病系)作为材料,利用cDNA-AFLP技术,研究了花椰菜黑腐病抗性在黑腐病菌侵染和非侵染条件下基因表达的情况.获得了两个阳性克隆M2和M6.Nprthern杂交和点杂交进一步证明M2是组成型表达的cDNA片段,只是在病菌侵染与非侵染条件下表达丰度不同.而M6是差异表达的cDNA片段.Southern杂交表明M6 cDNA片段在花椰菜基因组中是单拷贝序列.随后的序列分析发现M6 cDNA片段与拟南芥1号染色体的BAC F19P19中66 665～66 813 bp有84%同源性,该片段编码拟南芥的2A6蛋白的部分序列.推测的氨基酸序列与拟南芥的2A6蛋白部分序列有91%同源性.用H2O2作为外源分子胁迫处理的初步功能分析发现:M6 cDNA片段受H2O2诱导,在诱导早期16～24 h高度表达,其在叶片中的积累呈逐渐上升趋势.根据序列分析和初步的功能分析的结果推测:M6 cDNA片段可能就是编码花椰菜中类2A6蛋白的部分基因片段.是参与花椰菜抗病反应信号途径的相关调控基因片段.     

蝴蝶兰查尔酮合酶基因cDNA的克隆、鉴定及其原核表达

利用逆转录．聚合酶链式反应(RT-PCR)得到在蝴蝶兰花瓣中特异性表达的查尔酮合酶cDNA(pchs-1)．DNA序列分析表明,该序列与已发表的查尔酮合酶基因有很高的同源性．将此cDNA序列不改变阅读框架克隆到pET-24α( )原核表达载体上,经IPTG诱导后,SDS-PAGE的结果表明有预测大小的蛋白受诱导表达,该基因在植物中的功能验证工作正在进行中．     

稻瘟病抗性基因Pita~2的克隆及其介导的防御相关基因表达分析

为了克隆位于水稻12号染色体着丝粒区域具有广谱抗性的稻瘟病抗性基因Pita~2,研究构建并筛选了该基因供体品种PiNo.4的Fosmid文库,构建Pita~2候选区域的物理图谱并互补验证了5个候选基因,克隆得到了稻瘟病抗性基因Pita~2,确认了该基因就是Ptr基因.一组基因型接近、对Pita~2致病性相反的稻瘟病菌菌株对水稻进行离体接种,接种前后相关防御基因的表达量分析结果表明,Pita~2基因在稻瘟病菌侵染后均成功激活了水杨酸和茉莉酸抗病信号通路,主要通过参与水稻水杨酸信号通路赋予水稻抗性,该研究为利用Pita~2培育持久广谱抗性水稻提供了一定的理论基础.     

Construction and characterization of a normalized whole-life-cycle cDNA library of rice

A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies.For studying the functions of rice genes on a large scale,a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63,an elite restorer line for a number of rice hybrids that are widely cultivated in China,This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages.For normalization,the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library.This library consists of 62000 clones with an average insert length about 1.4kb.Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes.Sequencing of 10750 cDNA clones of this library reveals 6399 unique EST s(expressed sequence tags),indicating that the non-redundancy of the library is about 59.5%.This library has been used to make cDNA microarrays for functional genomic studies.     

Identification of genes associated with cotyledon senescence in upland cotton

Leaf senescence in plants is an essential develop- mental phase, and an understanding of senescence is important not only for pure scientific reasons, but also for practical purposes. During the last decade, a number of senescence-associated genes (SAGs) …     

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50—400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of RCC.     

Construction of cDNA library from iron-deficiency induced maize roots and screening and identification of iron-stress gene Fdr3

To isolate Fe-deficient related (Fdr) genes, an expression cDNA library of 4.5×105 pfu/μg has been constructed from maize roots in iron-stress. 6 clones have been screened from the cDNA library by differential hybridization screening. It is proved that an Fdr3 cDNA clone expressed stronger under iron-deficient condition than under iron-sufficient one by Northern blot and Western blot.     

杜洛克与梅山猪大卵泡消减文库的构建

为了鉴定杜洛克猪相对于梅山猪在大卵泡中高表达的基因,本研究应用抑制性消减杂交技术成功构建了以梅山大卵泡c DNA为driver,杜洛克大卵泡c DNA为tester的消减c DNA文库。结果显示:以G3PDH和β-actin为指标检测文库的消减效率为25,从该文库中获取了350个有效的阳性克隆,PCR检测插入片段主要分布在150-750 bp。克隆测序得到74个有效的EST序列,GO分析表明主要与细胞信号、细胞结构、代谢、细胞分化、基因/蛋白质合成、细胞组织防护等功能相关。利用q PCR技术验证了ELTD1、Grb14、SNRPE、CSDE1、ALDH18A1、e IF4E、BMPR-IB等基因在梅山和杜洛克大卵泡中的表达模式。结果发现在两猪种的大卵泡间存在显著性差异。本研究有助于揭示影响猪卵泡发育和生殖数量控制的分子基础。     

Identification of the candidate genes associated with cellular rejection in pig-to-human xenotransplantation

To identify the genes associated with cellular rejection in pig-to-human xenotransplantation, the suppression subtractive hybridization (SSH) was used in screening the up-regulated genes from a co-culture of human peripheral blood mononuclear cells (PBMCs) and porcine vascular endothelial cell line PIEC. The up-regulated cDNAs were cloned into pGEM-T Easy vector and then sequenced. Nucleic acid homology searches were performed using the BLAST program. A subtracted cDNA library including about 300 clones with the expected up-regulated genes was obtained. Twenty-four of these clones were analyzed by sequencing and homology comparison was made. These clones represent the genes of human perforin (PRF1), proteasome, lymphocyte specific interferon regulatory factor/interferon regulatory factor 4 (LSIRF/IRF 4), muscleblind-like (MBNL) protein and a porcine expressed sequence tag (EST) which has 81% homology with human oxidative-stress responsive 1 (OSR 1). These genes might be the candidate genes which are associated with cellular rejection in pig-to-human xenotransplantation.     

Construction of cDNA library from iron-deficiency induced maize roots and screening and identification of iron-stress geneFdr3

To isolate Fe-deficient related (Fdr) genes, an expression cDNA library of 4.5×10⁵ pfu/μg has been constructed from maize roots in iron-stress. 6 clones have been screened from the cDNA library by differential hybridization screening. It is proved that anFdr3 cDNA clone expressed stronger under iron-deficient condition than under iron-sufficient one by Northern blot and Western blot.     

Identification of differentially expressed genes in esophageal cancer through SSH in combination with high throughput reverse Northern screening

To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.