连接肽在多基因转化中的应用

多基因转化是当今遗传转化研究的热点之一,在植物基因工程中利用有自我剪切功能的口蹄疫病毒片段2A和凤仙花种子的一段多肽LP4作为连接肽进行多基因融合是一种可以替代传统多基因转化方法的新手段,连接肽可以同时连接多个基因并且保证多基因的协同表达,将LP4和2A多肽杂合形成的新的多肽(LP4/2A)兼具两者优势,是一种更为有效的多基因转化策略。     

以耐草甘膦2mG2-epsps基因为选择标记的玉米转化体系的建立

转化细胞的筛选和再生是植物遗传转化体系中重要的组成部分,筛选剂的选择和筛选压力的高低直接影响着外源基因的转化率。以草甘膦异丙胺盐为筛选剂,通过比较在不同的草甘膦浓度及筛选时间的条件下,玉米愈伤组织生长和分化情况,发现经1mM的草甘膦异丙胺盐筛选15d后玉米愈伤组织分化受到明显抑制,故以此作为玉米遗传转化实验中的筛选压力。通过基因枪轰击,将构建好的pMAGUHM载体（其上携带有抗草甘膦基因2mG2-epsps基因）转化到玉米愈伤组织,利用草甘膦筛选得到耐草甘膦植株80株,其中PCR检测阳性植株为36株,转化率为45％。     

融合基因在转基因植物中的应用

目前双基因和多基因转基因植物已经商品化,并展现了广泛的应用前景。但在转基因植物研究中,使多个基因同时在植物体中表达调控依然很难实现,是植物基因工程和生物技术发展中的难点。融合基因表达载体作为一种新型的方法,弥补了获得双价或多价转基因植物传统方法的缺点,具有更高的应用价值。本文对目前构建融合基因的方法作了评述,并对比较新颖的连接肽2A和LP4做了详细介绍。     

含VHb基因和GFMcryIA基因的双价基因植物高效表达载体的构建

将人工合成的、密码子优化后的透明颤菌血红蛋白基因与人工合成的GFMcryIA基因构建成双价基因植物高效表达载体PGBI4ASVHBBt,vgbM基因表达盒中含2个增强子的35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子;Bt基因表达盒中,除含有以上提高转录和翻译的调控元件外,还包含有正确切割、加工序列、Poly(A)信号序列。利用根癌农杆菌介导转化烟草,获得了转基因植株;PCR及Southern blot检测,证实了双价基因在烟草基因组中的整合;Western blot检测证实了vgbM基因在转基因烟草中的表达;杀虫实验表明GFMcryIA基因也表达出活性毒蛋白。     

转Bt cry1Ah基因抗虫玉米的分子检测及农艺性状分析

利用农杆菌介导法将具有自主知识产权的Bt cry1Ah基因转入玉米自交系综31中,在获得的1 764株转基因植株中筛选获得1株对玉米螟有显著抗性的转基因植株。对该抗性植株进行了室内和田间抗虫性分析及外源基因表达分析,并研究了其杂交后代的农艺性状。结果表明,该转基因植株对亚洲玉米螟田间抗虫性达到高抗水平;外源基因能够正常高效表达,在玉米抽穗期的叶片中Cry1Ah蛋白表达量最高,达到1μg/g鲜重;株高、穗位高、穗长、穗粗等与非转基因对照植株相比差异不大,秃尖长明显短于对照,千粒重及单株产量明显高于对照,这种差异在人工模拟虫害爆发情况下达到极显著水平。     

转双抗虫基因烟草的研究

用改造的雪花莲凝集素基因GNAmm与合成的苏云金芽孢杆菌(Bt)毒蛋白cry1Ac基因构建了带有双价基因的植物表达载体,在该表达载体中这两个基因的转录分别受笋瓜PP2启动子(SPP2P)和CaMV 35S启动子的调控。通过根癌土壤杆菌介导转化法,获得了一批抗卡那霉素的转化再生烟草植株。PCR检测及基因组DNA Southern blot\,Slot blot杂交分析的结果表明Gna基因和Bt基因已整合到烟草总DNA中。用Bt毒蛋白抗血清进行Western blot分析,转基因植株均有Bt杀虫蛋白的不同程度的表达。对转化再生烟草的虫试结果表明,在所受试的19株烟草中60%的植株上的棉铃虫在5天内死亡率达到100%,而且存活幼虫的生长发育受到明显抑制;蚜虫抑制生长试验表明,多数转化再生植株具有较强的抗蚜活性,平均能够抑制桃蚜50%～60%的蚜口密度,有的高达80%以上。以上结果表明利用这两个改造过的抗虫基因可以获得既抗虫又耐蚜的转双抗虫转基因植物。     

苏云金芽胞杆菌高效表达载体的构建

[目的]通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性,筛选出一个强启动子,利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)高效表达载体.[方法]利用启动子融合lacZ技术检测了4种启动子的转录活性.通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证.[结果]构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E4个启动子融合报告基因lacZ的表达载体,经β-半乳糖苷酶活性分析得知,启动子活性从高到低依次为Pcry8E＞Pcry1A＞Pcry4A＞Pcry3A.选取cry8E启动子,以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b,将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上,分别转入无晶体突变株HD-73-,获得菌株HD-8E1Ac和HD-422-1Ac.扫描电子显微镜观察显示,HD-8E1Ac菌株可以形成菱形晶体,说明正确表达了cry1Ac基因.SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b.对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性,且菌株活性高于HD-422-1Ac.[结论]利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b,该载体可正确表达cry1Ac基因,其表达效率高于被广泛应用的pSXY422b.     

苏云金芽孢杆菌cry2Aa操纵元蛋白对杀虫蛋白晶体形成作用的研究

利用重叠PCR的方法,通过两次PCR扩增,分别获得cry2A10操纵元的orf1、orf1 orf2与cry2Ab5基因的融合片段。融合片段经BamHⅠ和EcoRⅠ双酶切与pHT315连接,分别构建了基因融合片段的原核表达载体pFU(orf1 2Ab)和pFU(orf1 orf2 2Ab),电转化Bt无晶体突变株4Q7后,扫描电镜下可观察到典型的方形晶体,通过SDS-PAGE可检测到60kD大小的蛋白表达带。结果表明,cry2Ab5可在cry2a0的启动子帮助下有效转录和表达,并在orf2产物帮助下形成蛋白晶体。     

人干扰素α-2b 基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b（Human interferon α-2b,HuIFN α-2b）编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

苏云金芽孢杆菌B-Pr-88菌株中cry2Ab4基因的表达和杀虫活性研究

以我室自行分离的对鳞翅目夜蛾科害虫具有高毒力的Bt菌株B-Pr-88为材料,用PCR-RFLP方法从其质粒DNA文库中筛选到含cry2Ab基因的一个阳性克隆pZF858,序列测定发现,该片段含有cry2Ab全长基因,开放读码框为1902bps,编码由633个氨基酸组成的70.7kD蛋白,氨基酸同源性与已公布的cry2Ab基因同源性均为99.8%,经Bt基因国际命名委员会正式命名为cry2Ab4。根据cry2Ab4基因开放阅读框架(ORF)两端序列,设计合成一对特异引物L2ab5和L2ab3,PCR扩增获得cry2Ab4完整ORF,与大肠杆菌表达载体pET-21b连接,构建了重组表达质粒pET-2Ab4,质粒导入大肠杆菌BL21(DE3),IPTG诱导后,SDS-PAGE电泳证实该基因表达了60kD的蛋白,生物测定表明,Cry2Ab4对棉铃虫和大豆食心虫具有高毒力,同时对小菜蛾和二化螟有一定的杀虫活性,而对亚洲玉米螟和甜菜夜蛾没有杀虫活性。     

Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation

The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG ( 2 ) -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG(2), were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.     

Protein restriction to pregnant rats increases the plasma levels of angiotensin II and expression of angiotensin II receptors in uterine arteries

Whether gestational protein restriction affects the renin-angiotensin system (RAS) in uterine artery remains unknown. In this study, we hypothesized that gestational protein restriction alters the expression of RAS components in uterine artery. In study one, time-scheduled pregnant Sprague Dawley rats were fed a normal or low-protein (LP) diet from Day 3 of pregnancy until they were killed at Days 19 and 22. The uterine arteries were collected and used for gene expression of Ace, Ace2, Agtr1a, Agtr1b, Agtr2, Esr1, and Esr2 by quantitative real-time PCR and/or Western blotting. LP increased plasma levels of angiotensin II in pregnant rats. In the uterine artery, the expressions of Agtr1a, Agtr1b, and Esr1 were increased by LP at Days 19 and 22 of pregnancy, whereas the abundance of AGTR1 and AGTR2 was increased by LP at Day 19 of pregnancy. The expression of Ace2 was not detectable in rat uterine artery. In study two, virgin female rats were ovariectomized and implanted with either 17beta-estradiol (E2), progesterone (P4), both E2 and P4, or placebo pellets until they were killed 7 days later. In rat uterine artery, E2 and P4 reduced the expression of Agtr1a, and E2 increased the expression of Agtr1b and Agtr2, but neither E2 nor P4 regulated the expression of Ace. These results indicate that gestational protein restriction induces an increase in Agtr1 expression in uterine artery, and thus may exacerbate the vasoconstriction to elevated angiotensin II present in maternal circulation, and that female sex hormones also play a role in this process.     

Differential gene expression of TRPM1, the potential cause of congenital stationary night blindness and coat spotting patterns (LP) in the Appaloosa horse (Equus caballus)

The appaloosa coat spotting pattern in horses is caused by a single incomplete dominant gene (LP). Homozygosity for LP (LP/LP) is directly associated with congenital stationary night blindness (CSNB) in Appaloosa horses. LP maps to a 6-cM region on ECA1. We investigated the relative expression of two functional candidate genes located in this LP candidate region (TRPM1 and OCA2), as well as three other linked loci (TJP1, MTMR10, and OTUD7A) by quantitative real-time RT-PCR. No large differences were found for expression levels of TJP1, MTMR10, OTUD7A, and OCA2. However, TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) expression in the retina of homozygous appaloosa horses was 0.05% the level found in non-appaloosa horses (R = 0.0005). This constitutes a >1800-fold change (FC) decrease in TRPM1 gene expression in the retina (FC = -1870.637, P = 0.001) of CSNB-affected (LP/LP) horses. TRPM1 was also downregulated in LP/LP pigmented skin (R = 0.005, FC = -193.963, P = 0.001) and in LP/LP unpigmented skin (R = 0.003, FC = -288.686, P = 0.001) and was downregulated to a lesser extent in LP/lp unpigmented skin (R = 0.027, FC = -36.583, P = 0.001). TRP proteins are thought to have a role in controlling intracellular Ca(2+) concentration. Decreased expression of TRPM1 in the eye and the skin may alter bipolar cell signaling as well as melanocyte function, thus causing both CSNB and LP in horses.     

LSY-2 is essential for maintaining the germ-soma distinction in C. elegans

The mechanisms that specify and maintain the characteristics of germ cells during animal development are poorly understood. In this study, we demonstrated that loss of function of the zinc-finger gene lsy-2 results in various somatic cells adopting germ cells characteristics, including expression of germline-specific P granules, enhanced RNAi activity and transgene silencing. The soma to germ transformation in lsy-2 mutants requires the activities of multiple chromatin remodeling complexes, including the MES-4 complex and the ISW-1 complex. The distinct germline-specific features in somatic cells and the gene expression profile indicate that LSY-2 acts in the Mec complex in this process. Our study demonstrated that lsy-2 functions in the maintenance of the soma-germ distinction.