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.     

家蚕基因组P450基因家族的分析

利用家蚕基因组mRNA和氨基酸数据库对P450基因进行搜索．结果显示：在家蚕基因组中发现77个P450基因．分别归于18个P450家族和26个亚家族,其中有9个多基因家族。3个基因家族的成员超过10个,CYP4有20个成员。是一个典型的多基因家族。对P450基因进行正选择、基因转换和基序分析。结果表明：1个组（包含3个P450s基因）中检测出了显著的正选择替换位点,在这1个组中．转换事件的次数为3次。     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

细胞色素P450与农药相互作用及其机理研究

综述了细胞色素P450的种类和功能多样性,介绍了细胞色素P450参与除草剂代谢及其作用机理．同时,简述了细胞色素P450 14DM与杀菌剂特异性作用的机制．报道了我们从抗除草剂的瑞士黑麦草中克隆CYP8181同源基因及其功能的研究,以及柑橘绿霉菌细胞色素P450 14DM基因的克隆表达．为深入研究杀菌剂作用细胞色素P450 14DM的机理从而设计以P450 14DM为特异性靶标的新型农药、以及进一步研究细胞色素P450酶系与除草剂代谢的关系打下了良好的基础．     

Identification and functional analysis of 23 novel box C／D snoRNAs from Oryza sativa

In a cDNA library generated from rice small nuclear RNAs,30box C/D small nucleolar RNAs (snoRNAs) were identiffied through preliminary screen.Except 7 known snoRNAs such as U14,all snoRNAs were identified in rice for the first time experimentally.Among the 23 novel snoRNAs,11 snoRNAs appear rice-specific,6 snoRNAs are unique to plants,the remaining 6 snoRNAs have their counterparts in both Arabidopsis and yeast or mammals according to the conserved antisense sequencs that guide 2‘-O-ribose methylation of rRNA,17 of the 23 novel snoRNAs were predicted to guide 24 2‘‘-O-ribose methylations at the specificsites of rice 5.8S,18S,25S rRNAs,among which 19 methylated sites were determined by primer extension at low dNTP concentrations.The remaining 6 snoRNAs devoid of rRNA antisense elements may represent novel snoRNA species in rice.The results show that constructing a cDNA library from small nuclear RNAs is an effective experimental approach for novel snoRNA is identification.The novel snoRNAs are important in elucidating the genomic organization and expression of plant snoRNA genes and the mechanism through which 2‘‘-O-ribose methylations took place in rRNAs.     

Two closely linked rice U14 boxC/D snoRNAs

By analysis of the conserved elements in yeast U14 boxC/D snoRNA. the conserved elements in rice U14 boxC/D snoRNA have been speculated. Through computer search of the international rice genome database, two rice U14 snoRNA gene candidates are obtained. These two putative U14 snoRNA genes are closely linked on rice chromosome 2. The coding sequences of these two snoR-NAs exhibit the hallmark structure of boxC/D antisense snoRNA. They both have conserved boxC and boxD sequences and a 14nt-long complement to the sequence between 414nt and 427nt of rice 18S rRNA (according to GenBank accession no. X00755). The experimental evidence shows that these two snoRNAs are involved in the methylation of the complementary sequence of rice 18S rRNA. The existence and localization of these two snoRNAs are proved by RT-PCR and Northern blot. Further analysis shows that both of the newly found rice snoRNAs have high homology with maize U14 snoRNA. and they are named rice U14.1 snoRNA and U14.2 snoRNA respectively. The gene sequence encoding these two snoRNAs has been deposited in the GenBank database under accession number of AF332622.     

亚洲棉5号染色体RGAs克隆与分析

棉花是最主要的天然纤维作物,深入进行棉花基因组研究具有重要意义.采用酶解、前后低渗和轻压相结合的方法制备亚洲棉染色体中期膜载片,激光法分离亚洲棉第5号单染色体,建单染色体扩增池后克隆其抗病基因同源序列(RGAs),获得P7、P12、P19和P23等4个序列.序列比对和聚类分析表明,这4条序列均为NBS-LRR类RGAs,P7、P12、P19聚成一类,它们之间的同源性很高,P23聚成另一类,与黑松的RPS2和油菜的RGA30同源性较高.为该染色体分子标记开发、基因克隆乃至全序列测定奠定基础.     

一个与人类待定肿瘤抑制基因同源的水稻基因的分离

从水稻基因文库中筛选到一个与人类待定肿瘤抑制基因QM同源的基因,命名为OSQM2．该基因片断长为3．1kb,编码一个具有184个氨基酸的高度碱性的蛋白质,与其他已知QM基因相比,该基因具有一个非常特殊的启动子,包含许多在植物中已发现的与抗逆有关的顺式作用因子,如：“G盒”,“DRE盒”,“MYC盒”等,故而该基因有可能是一个新的受环境胁迫因子诱导的基因,Southern杂交表明它以单拷贝形式存在于基因组中。     

用RT-PCR技术研究水稻中抗病基因同源序列DFR-1、OsMlo-1所在基因的功能

DFR-1、OsMlo-1分别是最近从水稻中克隆的玉米Hml和大麦中Mlo抗病基因同源序列，这两个序列与两个已报道的水稻抗稻瘟病数量性状位点（QTLs）有较好的对应关系，表明它们所在的基因可能参与抗病反应，为了进一步研究水稻DFR-1、OsMlo-1所在基因的功能，在DFR-1、OsMlo-1假定的外显子上设计引物，通过RT-PCR技术，研究在接种白叶枯病（Xanthomanas oryza pv oryzae）菌株PX099以及接种稻瘟病（Magnoparthe grisea）菌株V86013前后，水稻品种IRBB13和水稻品种明恢63中DFR-1、OsMlo-1所在基因的表达．结果表明：在接种白叶枯病菌株PX099的水稻品种IRBBl3中与DFR-1对应的基因是诱导增强的；在接种稻瘟病菌株V86013的水稻品种明恢63中与DFR-1、OsMlo-1对应的基因是诱导增强的，进一步表明DFR-1、OsMlo-1所在的基因可能参与水稻抗病反应。     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that t...     

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (Crucian carp (Carassius auratus L.) blastulae )cells,and thus defend host cells against the virus invasion ,The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes,In this study ,suppressive subtractive hybridization is applied to constructing a subtracted ,cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells,272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library .Sequencing analysis reveals 69 genes,including 46 known gene homlologues,and 23 unknown putative genes,The known genes include the gemes involved in interferon signaling pathways,such as Stat1 and Jak1,the antiviral gences,such as Mx and Vipering,and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknow putative genes contain AU-rich element in their sequences,Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR,The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells,but also leads to the expression of a series of antiviral-relevant genes or immune-releveant genes,and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.     

Molecular cloning, chromosomal mapping and expression analysis of disease resistance gene homologues in rice (Oryza sativa L.)

Resistance-like sequences have been amplified from first strand cDNA and genomic DNA of rice by PCR using oligonucleotide primers designed from sequence motifs conserved between resistance genes of tobacco andArabidopsis thaliana. 3 PCR clones, designatedOsr1, Osr2 andOsr3 which were 98% identical in nucleotide sequence level, have been found to be significantly homologous to known plant resistance genes and all contained the conserved motifs of NBS-LRR type resistance genes, such as P-loop, kinase2a, kinase3a and transmembrane domain.Southern hybridization revealed that rice resistance gene hornologueswere organized as a cluster in the genome. RFLP mapping using a DH population derived from anindica/japonka cross (Zhaiyeqing 8/Jingxi 17) and an RFLP linkage map assigned two copies ofOsrl and one copy ofOsr3 to the distal position of chromosome 12 where a blast resistance QTL has been mapped previously. Northern blot analysis showed thatOsrl gene was constitutively transcribed in rice leaves, shoots and roots. Further study concerning isolation of full-length cDNAs would be conducive to elucidating the functions of these genes.     

生物信息学技术克隆并分析新基因STRF7

为进一步研究信号转导相关的新基因片段BE644250,采用生物信息学方法克隆基全长cDNA,并分析了其ORF,电子表达谱,染色体定位等,之后对全长序列进行了实验验证。电子延伸（contig)获得了729bp的延伸产物,含一个典型的74aa的ORF,命名为STRF7。与已知蛋白无明显同源性,部分地相似于人的源框蛋白CDX－4和酵母的转录调节子ADR6,属一新发现的基因;RT－PCR从IL－6刺激后的U937中克隆了STRF7基因,基序列与电子延伸结果安全一致,进一步的分析显示STRF7在多种组织中表达并定位于第6号染色体上,上述结果显示,STRF7是一个新基因,编码含74aa的蛋白,并且是一个潜在的转录因子。     

从EST出发克隆人基因组Xq13区段一个候选基因的初步研究

人类基因组表达序列筛选是寻找候选基因的重要路线之一，外显子陷阱法，ｃＤＮＡ直接筛筛选法，它们可分别根据表达序列的结构及表达特点进行筛选，ＥＳＴ是表达图的位标，它们是一些位点专一的表达序列位标，根据ＥＳＴ的特征，在国内首次建立了一种从ＥＳＴ出发的筛选候选基因的新方法，用睦方法已在人Ｘ染色体Ｘｑ１３区段筛选得到了一个新的ｃＤＮＡ，总测序徇的１３９８ｂｐ包含了完整的３末端。     

3个白桦细胞色素P450基因生物信息学及表达分析

【目的】研究白桦细胞色素P450基因表达的组织特异性,以及在茉莉酸甲酯(MeJA)、水杨酸(SA)、赤霉素(GA₃)、脱落酸(ABA)、乙烯利和伤害处理下的基因表达模式。【方法】筛选白桦转录组,获得3个BpCYP450 基因,分别命名为BpCYP4、BpCYP5、 BpCYP14,利用生物信息学分析BpCYP450蛋白的分子结构特征及其与其他物种CYP450蛋白的亲缘关系。采用QRT-PCR技术,对白桦BpCYP450组织特异性、激素信号及伤害诱导下的表达特征进行分析。【结果】生物信息学结果表明BpCYP4、BpCYP5、BpCYP14 cDNA序列长度分别为1 569、1 584和1 530 bp,具有完整的开放阅读框(ORF),分别编码522、527和509个氨基酸,均为亲水性跨膜蛋白,主要定位于叶绿体。白桦BpCYP450与百脉根、扁豆及豌豆等豆科植物CYP450蛋白亲缘性较高。组织特异性分析结果显示,3个BpCYP450 基因在叶和根中表达较高,茎中较低。激素信号及伤害诱导结果表明,3个BpCYP450基因不同程度地响应MeJA、SA、GA₃、ABA、乙烯利激素信号及伤害诱导。【结论】3个BpCYP450基因均为CYP450基因家族的新成员,具有组织特异性及激素诱导表达特性,可能在白桦生长发育、抵御胁迫及代谢物合成中发挥重要作用。     

Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus

~~Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus~~