转新型抗虫基因Vip3A棉花新种质的创制

该研究构建植物表达载体pBin438 Vip3A,通过农杆菌介导法转化棉花品种‘冀合713’,将新型抗虫基因Vip3A导入到棉花植株,创制对棉铃虫抗性的转基因棉花新种质。结果表明：(1)PCR检测Vip3A基因已经导入到棉花基因组中且能够稳定遗传。(2)室内抗虫性鉴定表明,与对照相比转基因植株对棉铃虫的抗性显著提高,并获得2株高抗和3株抗棉铃虫的转Vip3A基因株系。(3)Southern blotting结果显示,转基因株系BV01为单拷贝。(4)Elisa检测表明,外源Vip3A基因在BV01的根、茎、叶、花、种子中都有表达,在叶片中的Vip3A蛋白表达为苗期>蕾期>花期>铃期>吐絮期。该研究创制了新型抗虫转基因棉花材料,为培育棉花新型抗虫品种提供了种质资源。     

海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

沉默大豆GmWRKY33B基因导致大豆抗病性降低

WRKY转录因子基因家族是植物特有的转录因子,在防御中起着重要作用。通过生物信息学分析,本研究在古四倍体大豆(Glycine max)基因组中找到一对同源性高达93%的WRKY33同源基因,并将其命名为GmWRKY33B。从GmWRKY33B的两个同源基因保守区域选取一个315 bp片段构建至菜豆豆荚斑驳病毒(bean pod mosaic virus, BPMV)沉默载体(BPMV-VIGS)上,以期同时沉默上述2个GmWRKY33B基因。结果表明,同时沉默2个GmWRKY33B基因并不显著改变沉默植株的表型,但却显著降低了大豆对大豆斑点病菌以及大豆花叶病毒的抗性,说明GmWRKY33B在大豆免疫反应中起正调控作用。激酶分析表明,GmWRKY33B沉默植株中flg22诱导的GmMPK6的磷酸化水平较空载体BPMV-0植株显著降低,说明GmWRKY33B可以通过调控GmMPK6的激酶活性而参与大豆的免疫反应。抗毒素为大豆中主要起防御作用的植保素,而大豆异黄酮类特异性异戊烯基转移酶(prenyltransferase, PT)基因家族是参与大豆抗毒素生物合成的主要基因,许多PT基因启动子区含有与WRKY特异性结合的W-box序列。在丁香假单胞菌pv.甘氨酸(Pseudomonas syringae pv. glycinea, Psg)侵染条件下,4个PT基因的表达水平在沉默株系中显著降低,说明GmWRKY33B参与PT基因的转录激活。综上所述,GmWRKY33B通过调控GmMPK6的激活以及调控大豆抗毒素生物合成途径中关键酶编码基因的表达而参与免疫反应。     

沙棘hrh miR319e靶向转录因子AP4调控种子发育的研究

为探讨沙棘hrh miR319e与转录因子AP4间的靶向关系,并分析其在种子发育过程中的表达,以不同发育期的种子为材料,运用RNAhybrid软件预测hrh miR319e成熟体序列与其候选靶基因AP4的3′ UTR区的结合位点,构建pcDNA3.1+pmirGLO AP4和pcDNA3.1 hrh mir319e+pmirGLO AP4载体,用双荧光素酶报告基因检测方法验证hrh miR319e与候选靶基因AP4间的靶向关系,并采用qRT PCR方法分析hrh miR319e与AP4基因在沙棘不同发育期种子中的表达变化。结果显示：(1) 生物信息学预测AP4 3′ UTR区与hrh miR319e成熟体序列完全互补。(2) 荧光检测显示pcDNA3.1 hrh mir319e+pmirGLO AP4质粒显著抑制荧光素酶活性(P< 0.001)。(3) 不同发育期种子中hrh miR319e的表达量总体呈现出先明显上升后明显降低的变化趋势,而AP4基因的表达量则呈现出先降低后上升的趋势;当hrh miR319e的表达量在花后80 d达到最高水平时,‘新俄3号’种子中为3.146、‘绥棘1号’种子中为4.298、‘杂56’种子中为3.892,AP4的表达量则为种子不同发育期的最低值,‘新俄3号’中为0.427、‘绥棘1号’中为0.526、‘杂56’中为0.451。研究表明,沙棘AP4是hrh miR319e的靶基因,hrh miR319e AP4模块在沙棘种子发育过程中存在负调控关系。该研究结果为深入了解沙棘种子发育机制及大粒品种培育提供了理论依据。     

棉花肉桂醇脱氢酶基因在棉纤维中的表达及对其结构组分的影响

利用农杆菌介导法获得‘新陆早36号’转棉花肉桂醇脱氢酶基因(GhCAD6)材料,以转基因T₆代植株为试材,对GhCAD6基因在叶片基因组中的整合情况和不同发育阶段棉花纤维中的表达量进行分析,研究该基因对棉纤维中结构多糖和苯丙烷类化合物含量及纤维中苯丙烷类结构单体的影响。结果显示：(1)GhCAD6基因以单拷贝的形式整合到受体棉花基因组中。(2)转基因植株纤维中GhCAD6基因的表达量低于相同发育阶段对照样品,在对照样品中GhCAD6基因的表达量表现为先升高,在20 DPA(开花后天数)时表达量最高,之后下降,而在转基因植株纤维中先上升,并于发育15 DPA时表达量下降,20 DPA时又上升至最高,之后再次下降。(3)成熟纤维中,转GhCAD6基因植株纤维中苯丙烷类化合物含量低于对照,但结构多糖含量的差别不明显。(4)转GhCAD6基因纤维中苯丙烷类结构单体——紫丁香基木质素(S 木质素)和愈疮木基木质素(G 木质素)的比值下降。研究表明,棉花转入GhCAD6基因后,纤维发育(15 DPA)中GhCAD6基因的表达量变化可能导致棉花中苯丙烷类化合物含量及其结构单体比率变化,从而造成棉花纤维品质改变。该研究结果可为深入分析GhCAD6基因在改良棉花纤维品质的作用机理提供理论依据。     

小麦盐华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达迫相关基因的克隆与表达分析

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。     

刺葡萄白藜芦醇合成酶基因对不同光质的响应及转录因子筛选

为探究白藜芦醇合成酶基因(RS)的表达模式和转录调控特征,该研究以刺葡萄愈伤组织为材料,采用RT PCR方法进行RS1基因克隆,分析RS1基因在8种不同光质培养条件下的表达模式,并对靶向RS1的转录因子进行预测分析和筛选验证。结果表明：(1)成功从刺葡萄中克隆获得RS1基因(GenBank登录号为OM339527);RS1基因开放阅读框为1 179 bp,由2个外显子和1个内含子组成,编码392个氨基酸,为亲水性无信号肽的细胞质定位蛋白,磷酸化修饰主要发生于苏氨酸和丝氨酸位点上,蛋白质的二级结构主要由α 螺旋、无规则卷曲和延伸链组成。(2)RS1启动子具有多个光响应和转录因子识别与结合元件,还涉及激素调控、生长发育、环境条件响应。(3)转录调控预测发现,靶向RS1的转录因子来自9个家族,共有23个成员,其中MYB和Dof基因家族具有多个成员和RS1启动子结合位点。(4)共线性分析表明,葡萄与毛果杨的共线性最高,MYB DIV和Dof5.1具有多个共线基因。(5)转录水平分析显示,长波光促进RS1的表达,在不同光质和培养阶段下MYB DIV与靶基因RS1具有相同的表达模式,而Dof5.1的表达模式与RS1呈相反趋势,表明MYB DIV和Dof5.1分别通过正负调控参与RS1的转录表达。     

芥蓝miR156a家族进化特性及表达分析

miRNA广泛参与植物的发育过程。芥蓝形态多样,叶片发育因品种而异。为了解miR156a家族的进化特性及其在芥蓝叶片发育中的表达模式。该研究对芥蓝miR156a成员及其前体pre miR156a成员进行生物信息学分析,比较不同品种芥蓝叶形的差异,并采用qRT PCR方法分析芥蓝不同组织部位中pre miR156a的表达水平、以及叶形相似的芥蓝品种‘翠宝’和‘改良香菇’中不同类型叶片的pre miR156a成员及其靶基因的表达水平。结果表明：(1)多序列比对和进化树分析发现芥蓝miR156a家族成员和pre miR156a 3p_1在进化过程中高度保守;二级结构预测发现pre miR156a每个成员均能形成茎环结构并包含2～3个miR156a成员序列;靶基因预测显示miR156a 5p和miR156a主要靶向SPL,而miR156a 3p_1则靶向CTPS等不同的基因。(2)‘翠宝’和‘改良香菇’芥蓝的叶形最为相似,qRT PCR分析显示,pre miR156a在‘翠宝’营养生长期的叶片中高度表达。(3)pre miR156a成员在‘翠宝’和‘改良香菇’不同类型叶片中差异表达,pre miR156a主要在成熟叶和菇叶中表达,而pre miR156a 3p_1和pre miR156a 5p在第一片真叶中表达量更高。(4)靶基因分析的结果在不同品种中呈现不同趋势,‘翠宝’成熟叶中SPL10/15高表达,SPL2表达量降低;‘改良香菇’中SPL10在成熟叶和菇叶中表达降低,SPL15在菇叶中高表达,SPL2在不同类型叶片中表达无差异。研究认为,miR156a成员及其靶基因SPL2/10/15可能参与调控芥蓝叶片发育,不同品种中作用的靶基因可能存在差异,从而导致了各品种芥蓝叶片发育的差异。     

辣椒CaWRKY14转录因子的生物学特性研究

WRKY转录因子是植物响应病原菌胁迫最重要的转录因子之一,且参与抗病反应及信号传导通路的调控。为研究辣椒WRKY基因的生物学特征,以辣椒高抗疫病材料CM334为试材,克隆获得响应疫霉菌诱导的转录因子CaWRKY14。生物信息学分析表明,该基因DNA全长2 530 bp,cDNA全长1 662 bp,含有5个内含子,编码553个氨基酸,含有1个WRKY保守结构域,属于Group Ⅱ(b)。实时荧光定量表达分析表明,CaWRKY14不仅受ABA和疫霉菌胁迫诱导表达,且表达量分别在12 h和24 h时达到峰值,分别是对照的8.54和8.04倍,同时也受高盐、热激和干旱胁迫诱导。利用VIGS技术对CaWRKY14转录因子进行沉默后发现,抗病材料CM334接种疫霉菌后趋于发病。研究表明,CaWRKY14基因在辣椒响应疫霉菌胁迫进程中可能发挥着重要作用。     

表皮葡萄球菌SarA对atlE、lipA和zinC基因表达调控的研究

表皮葡萄球菌 (Staphylococcus epidermids) 是一种条件致病菌，SarA(Staphylococcal accessory regulator A)是该菌中一个全局性调控因子，它控制着细胞中许多与毒性相关的基因表达. 报道了SarA在转录水平直接调控atlE、lipA和zinC基因的表达. RT-PCR和lacZ报告基因的分析结果显示，在表皮葡萄球菌ATCC35984中，SarA对atlE (自溶酶基因) 表达起负调控作用，而对lipA (脂肪酶基因) 和zinC (膜相关锌金属蛋白酶基因) 的表达则有正调控作用. 生物信息学分析表明，SarA控制atlE，lipA和zinC 3种基因表达可能是通过与被调控基因上游的特定DNA序列的结合来实现的，该DNA结合区保守并富含AT碱基. 根据已报道的金黄色葡萄球菌中SarA的结合位点序列，利用Omiga软件分析并推测了SarA结合atlE，lipA和zinC的可能区域. 基于SarA是一种多功能的毒素相关调控因子，结果提示，SarA能调控众多因子，可以作为防治表皮葡萄球菌感染的一个药物筛选靶点.     

PbrmiR397a regulates lignification during stone cell development in pear fruit

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

MiR397b regulates both lignin content and seed number in Arabidopsis via modulating a laccase involved in lignin biosynthesis

Plant laccase (LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of microRNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a microRNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b (OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b‐resistant laccase mRNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397‐mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin.     

Developmental control of Helicoverpa armigera by ingestion of bacteria expressing dsRNA targeting an arginine kinase gene

Cotton bollworm (Helicoverpa armigera) is a polyphagous pest that causes agricultural and commercial losses in many parts of the world. These losses are compounded by insecticide abuse, which leads to insecticide resistance as well as environmental and food pollution. RNA interference (RNAi) is a powerful tool used in gene functional research and RNAi-based pest control. In this study, arginine kinase (AK) of cotton bollworm was selected as the target gene, as it plays a critical role in cellular energy metabolism in invertebrates. Two fragments of the H. armigera AK gene (HarmAK) were cloned into the L4440 vector to express double-stranded RNA (dsRNA) in Escherichia coli (HT115). The effects of different factors on dsRNA stability and the effect of silencing HarmAK on cotton bollworm were subsequently investigated. Both AK gene and protein expression levels were significantly inhibited in larvae, and the peak cumulative mortality rate of 44.44% was recorded on day 5, after 2^nd instar larvae were exposed to the artificial diet coated with the engineered bacteria. The two dsRNAs (dsAK1 and dsAK2) also caused drastic reductions in body weight (38.43% and 17.37%, respectively), body length (26.73% and 11.23%, respectively) and pupation rate (48.89% and 42.95%, respectively) compared to the control on day 5. The development and morphology of the larvae, pupae and adults that fed on the dsAK1 and dsAK2 bacteria were significantly impaired, while the control was not. Thus, AK is a potential target gene for RNAi-mediated cotton bollworm control.     

Cotton bollworm resistance to Bt transgenic cotton: A case analysis

Cotton bollworm (Helicoverpa armigera) is one of the most serious insect pests of cotton. Transgenic cotton expressing Cry toxins derived from a soil bacterium, Bacillus thuringiensis (Bt), has been produced to target this pest. Bt cotton has been widely planted around the world, and this has resulted in efficient control of bollworm populations with reduced use of synthetic insecticides. However, evolution of resistance by this pest threatens the continued success of Bt cotton. To date, no field populations of bollworm have evolved significant levels of resistance; however, several laboratory-selected Cry-resistant strains of H. armigera have been obtained, which suggests that bollworm has the capacity to evolve resistance to Bt. The development of resistance to Bt is of great concern, and there is a vast body of research in this area aimed at ensuring the continued success of Bt cotton. Here, we review studies on the evolution of Bt resistance in H. armigera, focusing on the biochemical and molecular basis of Bt resistance. We also discuss resistance management strategies, and monitoring programs implemented in China, Australia, and India.     

Transgenic cotton expressing synthesized scorpion insect toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae

A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44–98%. The AaHIT gene could used therefore an alternative to Bt toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.     

Artificial miRNA-mediated silencing of ecdysone receptor (EcR) affects larval development and oogenesis in Helicoverpa armigera

The insect pests are real threat to farmers as they affect the crop yield to a great extent. The use of chemical pesticides for insect pest control has always been a matter of concern as they pollute the environment and are also harmful for human health. Bt (Bacillus thuringensis) technology helped the farmers to get rid of the insect pests, but experienced a major drawback due to the evolution of insects gaining resistance towards these toxins. Hence, alternative strategies are high on demand to control insect pests. RNA-based gene silencing is emerging as a potential tool to tackle with this problem. In this study, we have shown the use of artificial microRNA (amiRNA) to specifically target the ecdysone receptor (EcR) gene of Helicoverpa armigera (cotton bollworm), which attacks several important crops like cotton, tomato chickpea, pigeon pea, etc and causes huge yield losses. Insect let-7a precursor miRNA (pre-miRNA) backbone was used to replace the native miRNA with that of amiRNA. The precursor backbone carrying the 21 nucleotide amiRNA sequence targeting HaEcR was cloned in bacterial L4440 vector for in vitro insect feeding experiments. Larvae fed with Escherichia coli expressing amiRNA-HaEcR showed a reduction in the expression of target gene as well as genes involved in the ecdysone signaling pathway downstream to EcR and exhibited mortality and developmental defects. Stem-loop RT-PCR revealed the presence of amiRNA in the insect larvae after feeding bacteria expressing amiRNA-HaEcR, which was otherwise absent in controls. We also found a significant drop in the reproduction potential (oogenesis) of moths which emerged from treated larvae as compared to control. These results demonstrate the successful use of an insect pre-miRNA backbone to express amiRNA for gene silencing studies in insects. The method is cost effective and can be exploited as an efficient and alternative tool for insect pest management.