Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses

To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity of rPR8-NS(0028) might be associated with the lower virus titer and IFN-β level in the lungs of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028. In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous monitoring of their mammalian pathogenicity.     

艾纳香脱氢酶基因家族的生物信息学分析

采用RT-PCR和RACE（Rapid amplification of cDNA ends）技术,从艾纳香（Blumea balsamifera (L.) DC.）叶片中克隆到4条编码艾纳香脱氢酶（BbADH1、BbADH2、BbADH3、BbADH4）基因的cDNA序列,并对4条核苷酸及其编码的氨基酸序列进行生物信息学分析。结果显示:4条艾纳香脱氢酶序列开放阅读框均在900 bp左右,蛋白质等电点（pI）值在5.0~9.0之间,含量最多的氨基酸为亮氨酸（Leu）,最少的为色氨酸（Try）,具有明显的疏水区和亲水区,N端未发现信号肽,且无跨膜区;同源性比对结果显示,艾纳香BbADH蛋白与其他植物中ADH蛋白具有高度的相似性,且具有脱氢酶的特征功能域;系统发育分析表明,艾纳香（B. balsamifera (L.) DC.）BbADH1和BbADH3同处于一个分支,且与胡杨（Populus euphratica Oliv.）PeADH物种亲缘关系较近,而艾纳香BbADH4和BbADH2处于不同分支,且分别与葡萄（Vitis vinifera）VvADH和烟草（Nicotiana tabacum）NtADH物种亲缘关系较近。     

猪链球菌蛋白表面展示系统的构建及展示蛋白的初步观察

为构建猪链球菌(Streptococcus suis)蛋白表面展示系统,本研究通过序列分析,确定猪链球菌的LPxTG蛋白及其信号肽(SP)和胞壁锚定基序(CWA),通过PCR扩增Peno-SP、GFP、CWA的DNA片段并融合,构建强启动子Peno控制表达编码SP-GFP-CWA融合蛋白的DNA片段,将该重组DNA片段连接pSET2载体,获得蛋白表面展示质粒,转化猪链球菌,构建得到以GFP为报告蛋白的猪链球菌蛋白表面展示系统。结果显示,利用猪链球菌的10个LPxTG蛋白及其SP和CWA序列,构建了10个含有Peno-SP-GFP-CWA融合片段的重组pSET2表面蛋白展示质粒pSsPSD1至pSsPSD10,分别转化猪链球菌05ZYH33,PCR鉴定显示其中7个转化猪链球菌。采用western blot初步检测其展示蛋白,结果显示,7个转化阳性菌株均能有效表达GFP蛋白,以成熟GFP条带为指标,均表现出了一定的外源GFP表面展示水平,分别命名为SsPSD1、SsPSD2、SsPSD4、SsPSD7-SsPSD10,其中SsPSD1、SsPSD4、SsPSD8和SsPSD9表面展示水平相对较好,在猪链球菌表面展示外源蛋白方面具有很好的潜力。本研究首次尝试建立猪链球菌蛋白表面展示系统,为猪链球菌表面递呈外源蛋白或抗原提供了新的策略。     

3D-MOTIF Method and Its Application to Microscale Topography

The 2D motif method is a surface characterization method promising to separate roughness and waviness from a profile and is adopted by ISO,so is meaningful to extend this method to 3D.Vincent's watershed algorithm is employed for the generation of 3D-motif.A smallest area selecting criteria is proposed for the use of clearing the small motifs which are concerned as noise.As there are no manipulating factors as 2D-method,a multi-scale analysis is employed based on area and depth criteria,the use of depth criteria is to prevent the combination of two adjacent motifs if there is a significant peak on the border of them.Finally the surface of C(100) are analyzed by the presented method,the texture of this surface has been characterized.     

马尾松天然次生林抚育间伐试验研究

马尾松天然次生林的抚育间伐试验结果表明:不同间伐强度对林木单株的胸径和材积生长的影响存在着显著性差异,以保留密度0.9×115株/667 m2和1.0×115株/667 m2对胸径的生长影响效果最佳;但对林木的树高生长影响没有显著性差异。不同间伐强度对不同径阶胸径和材积生长的影响存在显著性差异,径阶越大其胸径和材积的生长就越快。不同间伐强度对林木蓄积量的影响也存在显著性差异,以保留密度1.1×115株/667 m2的蓄积量生长量最大,效果最为显著。     

淡色库蚊氯菊酯抗性相关基因PR-XP1 cDNA的克隆与序列分析

【目的】克隆淡色库蚊一个全新的氯菊酯抗性相关基因PR-XP1的全长序列,分析、预测其编码蛋白的结构与功能,为阐明该基因的抗性机制及研制新型卫生杀虫剂奠定基础。【方法】依据抗性与敏感品系库蚊差异表达的EST片段（GenBank号:EC093826.1）,设计特异性PCR引物,采用RACE技术,克隆淡色库蚊氯菊酯抗性相关基因PR-XP1的全长cDNA序列,运用生物信息学软件对其同源性与系统进化进行分析,并对PR-XP1编码蛋白的结构与功能进行预测。【结果】获得了全长为2 004bp的淡色库蚊氯菊酯抗性相关基因PR-XP1,该基因具有完整的CDS区,编码249个氨基酸。同源性比对与系统进化分析表明,PR-XP1基因核苷酸序列与致倦库蚊（GenBank号:XM₀01862469.1）、埃及伊蚊（GenBank号:XM₀01658032.1）和冈比亚按蚊（GenBank号:BX021208.1）相关基因的同源性分别为95%,83%和70%;其编码蛋白的氨基酸序列与致倦库蚊、埃及伊蚊、西方蜜蜂、入侵红火蚁、佛罗里达弓背蚁、印度跳蚁等昆虫中"假设性蛋白"的氨基酸序列同源性均达46%以上。结构分析显示,PR-XP1基因编码蛋白可能为具有2个跨膜螺旋的膜蛋白,跨膜区形成了一个特异性的"U"形结构;该基因还具有1个微弱的信号肽位点和24个磷酸化位点。【结论】克隆得到了一个全新的淡色库蚊氯菊酯抗性相关的"保守性假设蛋白"基因PR-XP1,推测其功能与氯菊酯抗性相关。     

鸡败血支原体pMGA多基因家族及其免疫逸机制的研究进展

pMGA多基因家族主要编码一类黏附素／血凝素蛋白，存在于鸡败血支原体的细胞表面，其主要功能是促进支原体黏附到宿主细胞上。近年来的相关研究发现，编码pMGA的基因数量从32－70不等，主要转录水平上通过（GAA）n 基序的数量改变引发pMGA基因的选择性转录，造成pMGA的抗原发生变异，从而干扰宿主正常免疫功能的发挥，使支原体对宿主产生严重的免疫逃逸。其中，（GAA）n基序已被证实在pMGA基因表达调控中起重要作用，这一三联体重复基序数目的多少直接影响到pMGA基因的ON／OFF不，本文通过对鸡败血支原体pMGA多基因家分子生物学研究进展浅要综述，以提出pMGA基因可能的转录调控机制，这对研究鸡败血支原体的分子致病机理及其免疫机制大有裨益。     

大豆基因组保守序列及综合保守序列扩增多态性分子标记(ICSAP)的设计

采用VBA程序查找了大豆基因组保守序列——在基因组中出现次数较多的碱基序列片段,分析了这类保守序列的基本特性,在此基础上,设计了一种新型分子标记——综合保守序列扩增多态性分子标记.碱基数目越多,保守序列种类数和出现次数越少,从10个碱基到27个碱基,保守序列的GC含量先是逐渐增加,达到一个平台后又逐渐降低.碱基离保守序列3＇端越近,出现A或T的可能性越大,碱基在保守序列中的分布具有非随机性.不同长度的保守序列具有明显的进化关系.筛选出10个碱基的保守序列和长度为8个碱基的填充序列用来设计引物,共得到566对引物组合.该新型分子标记有望在条带多态性、引物一致性等方面超越现有分子标记,同时具有成本低廉等优点.该研究旨在为基因组保守序列查找和分子标记设计方面提供新的思路,分析这些保守序列特性有助于进一步探索分子进化机理.该新型分子标记有可能在大豆种质资源鉴定和基因图位克隆等方面得到应用,同时该分析方法也适用对其他物种.     

P0 protein of cotton leafroll dwarf virus-atypical isolate is a weak RNA silencing suppressor and the avirulence determinant that breaks the cotton Cbd gene-based resistance

Cotton blue disease (CBD) is the most important disease present in cotton crops in South America and cotton leafroll dwarf virus (CLRDV) is the causal agent. The disease has been controlled by sowing cotton varieties resistant to CLRDV. However, in the 2009/10 growing season, an outbreak due to an atypical CLRDV isolate (CLRDV-at) occurred in northwest Argentina. Although CLRDV and CLRDV-at genomes are very closely related, the symptoms they produce in cotton plants are quite different. P0 is the most divergent protein between the isolates and in CLRDV is a silencing suppressor protein. This work characterized the silencing suppressor activity of the P0 protein encoded by CLRDV-at (P0^CL-at) and evaluated its role in Cbd-resistance break in cotton plants. It was demonstrated that P0^CL-at, despite having a mutation in the consensus of the F-box-like motif, was able to suppress local RNA silencing, but displayed lower activity than P0^CL. P0^CL and P0^CL-at showed no differences in the interaction with Gossypium hirsutum SKP1 orthologue (GSK1) and Nicotiana benthamiana SKP1 and both P0 proteins triggered destabilization of ARGONAUTE1. However, when the ability to enhance PVX symptoms was evaluated, P0^CL-at was shown to be a weaker pathogenicity factor than P0^CL in N. benthamiana. Interestingly, trans-expressed P0^CL-at enabled CLRDV to systemically infect CBD-resistant plants, and a chimeric CLRDV-P0^CL-at infectious clone succeeded in establishing infection in CBD-resistant cotton varieties with symptoms resembling those produced by CLRDV-at. These results strongly suggest that P0^CL-at is the avirulence (Avr) determinant involved in breaking cotton Cbd gene-based resistance.