当前位置: > 论文中心 > 科技论文 >

玉米ZmLTP3基因表达载体的构建及遗传转化

时间:2014-09-03 08:59 点击:
摘要:通过农杆菌介导转入拟南芥(Arabidopsis thaliana)中进行过量表达。PCR及Western-blot分析结果表明,ZmLTP3基因已转入拟南芥中,并且在大多数转化植株中稳定表达。 关键词:玉米(Zea mays);ZmLTP3基因;拟南芥(Arabidopsis thaliana);遗传转化
摘要:通过农杆菌介导转入拟南芥(Arabidopsis thaliana)中进行过量表达。PCR及Western-blot分析结果表明,ZmLTP3基因已转入拟南芥中,并且在大多数转化植株中稳定表达。
 
  关键词:玉米(Zea mays);ZmLTP3基因;拟南芥(Arabidopsis thaliana);遗传转化
 
  中图分类号:Q782 文献标识码:A 文章编号:0439-8114(2014)04-0929-03
 
  Construction and Transformation of Maize ZmLTP3 Gene Expression Vector
 
  SUN Xiao-yan,ZHU Yong,ZOU Hua-wen
 
  (College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China)
 
  Abstract: LTP3 plays important roles in plant physiology. To study the function of a LTP3-like maize(Zea mays) ZmLTP3 gene, the cDNA of ZmLTP3 was constructed into the expression vector pGreen0029 and over-expressed in Arabidopsis thaliana. PCR and Western-blot analysis showed that ZmLTP3 gene was transformed into A. thaliana and expressed in majority transgenic plants.
 
  Key words: maize(Zea mays); ZmLTP3 gene; Arabidopsis thaliana; transformation
 
  植物转脂蛋白(Lipid transfer proteins,LTPs)是一类小分子量、多基因家族编码的的碱性蛋白质,具备转移磷脂和脂肪酸的能力[1-3]。转脂蛋白分为两个亚家族,即LTP1和LTP2。LTP1亚家族成员含有90~95个氨基酸残基,分子质量为9 ku左右;LTP2亚家族成员通常含有70个氨基酸残基,分子质量为7 ku左右[4]。转脂蛋白家族成员的结构高度保守:分子内部含有两个保守的五肽基序(Thr/Ser-X1-X2-Asp-Arg/Lys、Pro-Tyr-X-Ile-Ser)。另外,蛋白质分子中都含有8个保守的半胱氨酸,形成4对二硫键[5];转脂蛋白分子还含有一个由4个α-螺旋形成的疏水洞穴,可以和所转运的磷脂或脂肪酸相互作用,从而行使其生物学功能[6]。
 
  目前为止,已经从多种植物中被克隆、鉴定了转脂蛋白基因[7-12]。除了拥有最初认为的脂转移功能外,还发现具有其他多种生物学功能,如种子脂肪动员、体细胞发育、蜡物质形成、表皮形成、花粉管黏着、抗病等[4,13-19]。相对于在生物胁迫中的功能而言,转脂蛋白在非生物胁迫中功能的直接证据还鲜有报道。
 
  在此前的研究中,课题组相继从玉米(Zea mays)中克隆了Pto/Pti1信号传导途径的两个重要成员ZmPto和ZmPti1基因,过量表达ZmPto和ZmPti1基因的拟南芥(Arabidopsis thaliana)株系表现出较高的抗盐性[20,21]。随后,对转基因拟南芥株系做基因芯片分析。结果表明,转基因拟南芥中LTP3基因(At5g59320)的表达量相对于野生型提高了近18倍。这表明LTP3可能是Pto/Pti1信号系统的下游组分,并且在抗盐过程中起到非常重要的作用。随后,从玉米中克隆了拟南芥LTP3基因的同源基因ZmLTP3(GenBank Accession No. JX435819),RT-PCR分析发现ZmLTP3可由多种生物和非生物胁迫因子(尤其是高盐)所诱导(待发表)。本研究构建了植物表达载体,将ZmLTP3基因转入拟南芥中过量表达,为研究其生物学功能提供可能。
 
  1 材料与方法
 
  1.1 试验材料
 
  1.1.1 材料 拟南芥(Columbia 生态型)、大肠杆菌(E. coli) DH5α、农杆菌GV3101、pGEM-T载体、含ZmPti1-dHA序列的酵母表达载体p426GAL1、含35S-C4DDPK-CBF3-NOS片段的植物表达载体pGreen0029均由长江大学农学院保存。
 
  1.1.2 试剂 DNA 凝胶回收试剂盒购自杭州V-gene生物技术公司; DNA限制性内切酶、T4 DNA 连接酶、ExTaq酶、DNA Marker均购自宝生物工程(大连)有限公司;Anti-HA(3f10)及Peroxidase-conjugated goat anti-rat IgG(H+L)均购自Roche公司;PVDF膜购自Millipore公司;其他试剂均为国产分析纯。PCR 引物及DNA测序工作由生工生物工程(上海)股份有限公司完成。
 
  1.2 试验方法
 
  1.2.1 ZmLTP3基因植物表达载体的构建 设计PCR引物(含BamHⅠ和StuⅠ酶切位点)扩增pGEM-T载体中ZmLTP3基因序列,BamHⅠ/StuⅠ双酶切PCR产物和含ZmPti1-dHA序列的酵母表达载体p426GAL1。T4 DNA连接酶连接酶切后的载体与PCR产物,形成重组载体p426GAL1。随后用BamHⅠ/PstⅠ双酶切重组载体p426GAL1和含有35S-C4DDPK-CBF3-NOS片段的植物表达载体pGreen0029,得到ZmLTP3-dHA片段和表达载体,T4 DNA连接酶连接过夜,形成含有35S-C4DDPK-ZmLTP3-dHA-NOS片段的重组载体pGreen0029,并送至生工生物工程(上海)股份有限公司测序验证。
 
  1.2.2 ZmLTP3基因的转化 将鉴定正确的质粒用冻融法转化农杆菌GV3101,转化后的农杆菌用花浸蘸法侵染拟南芥。
 
  1.2.3 转基因拟南芥的PCR检测 转基因拟南芥T0代的种子经消毒后播在含50 μg/mL卡那霉素的MS培养基上进行筛选,挑选在抗性培养基上正常生长的拟南芥植株提取基因组DNA,以DNA为模板,以ZmLTP3基因片断内部和载体片段的1对特异引物进行PCR鉴定。
 
  1.2.4 转基因拟南芥的Western-blot检测 取0.3 g经PCR鉴定呈阳性的拟南芥幼嫩的叶片,加0.5 mL蛋白提取缓冲液(10 mmol/L Tris-HCl,0.02% NaN3,0.001% PMSF,pH 8.0),液氮研磨后4 ℃、5 000 r/min离心10 min,吸取上清液,即得到总蛋白提取液。取适当总蛋白提取液电泳,转膜后以小鼠单克隆抗体Anti-HA(3f10)为一抗,Peroxidase-conjugated goat anti-rat IgG (H+L) 为二抗,进行Western-blot 检测。
 
  2 结果与分析
 
  2.1 ZmLTP3基因植物表达载体的构建
 
  取BamHⅠ/PstⅠ酶切回收后的PCR片段和载体片段, 用T4 DNA连接酶连接,转化大肠杆菌后,挑选阳性克隆提取质粒进行酶切验证,所得的酶切片段与预期大小一致, 初步证明已经连接成功。将质粒进行测序, 做进一步验证,结果确认了重组质粒构建成功。ZmLTP3重组质粒植物表达载体如图1所示。
 
  2.2 转基因拟南芥的鉴定
 
  经过测序鉴定正确的载体转化农杆菌,携带表达载体的农杆菌介导转化拟南芥, 转化后收取T0代种子, 播种在含有50 μg/L卡那霉素的MS培养基上。种子发芽后大多数幼苗子叶变黄,只有少部分幼苗仍然发绿,并且正常长出真叶,保持正常的生长状态,这部分很有可能就是转化了的幼苗(图2)。
 
  为了排除抗生素筛选过程中的假阳性必须对初步筛选出的抗性植株做进一步的分子检测。将在抗性培养基上正常生长的T1代幼苗转移到土壤中,待植株充分长大,有足够叶子时,取2~3片幼嫩的叶子提取基因组DNA,并以ZmLTP3基因与载体片段的特异引物进行PCR扩增,结果如图3所示。野生型植株没有扩增出相应条带,同时也发现,抗性筛选出的幼苗也有假阳性。
 
  为了进一步验证转基因株系,经过PCR 初步鉴定的株系,提取总蛋白,通过Western-blot进一步在蛋白水平检测基因的表达,结果如图4所示。除了野生型植株没有印迹条带外, 其他PCR 阳性的株系在13 ku左右处均有特异条带。结果表明, 这些株系为转基因株系,转化的目的基因在植物中得到了表达,没有出现基因沉默现象。
 
  3 小结
 
  参考文献:
 
  [1] BERNHARD W R, THOMA S, BOTELLA J, et al. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway[J]. Plant Physiol,1991,95(1):164-170.
 
  [2] KADER J C. Lipid transfer proteins in plants[J]. Annu Rev Plant Physiol Mol Biol,1996,47(1):627-654.
 
  [3] THOMA S, HECHT U, KIPPERS A,et al.Tissue specific expression of a gene encoding a cell wall localized lipid transfer protein from Arabidopsis[J]. Plant Physiol,1994,105(1):35-45.
 
  [4] CARVALHO A O, GOMES V M.Role of plant lipid transfer proteins in plant cell physiology: A concise review[J]. Peptide,2007,28(5):1144-1153.
 
  [5] DOULIEZ J P, MICHON T, ELMORJANI K, et al. Mini review: Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels[J]. Journal of Cereal Science,2000, 32(1):1-20.
 
  [6] ZACHOWSKI A, GUERBETTE F, GROSBOIS M,et al. Characterisation of acyl binding by a plant lipid-transfer protein[J]. European Journal of Biochemistry,1998,257(2):443-448.
 
  [7] KINLAW C S, GERTTULA S M, CARTER M C. Lipid transfer protein genes of loblolly pine are members of a complex gene family[J]. Plant Molecular Biology,1994,26(4):1213-1216.
 
  [8] FENG J X, JI S J, SHI Y H, et al. Analysis of five differentially expressed gene families in fast elongating cotton fiber[J]. Acta Biochimica et Biophysica Sinica,2004,36(1):51-56.
 
  [9] LIU K, JIANG H, MOORE S L, et al. Isolation and characterization of a lipid transfer protein expressed in ripening fruit of Capsicum chinense[J]. Planta,2006,223(4):672-683.
 
  [10] BOUTROT F, MEYNARD D, GUIDERDONI E, et al. The Triticum aestivum non-specific lipid transfer protein (TaLtp) gene family: Comparative promoter activity of six TaLtp genes in transgenic rice[J]. Planta,2007,225(4):843-862.
 
  [11] BOUTROT F, CHANTRET N, GAUTIER M F. Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein(nsLtp) gene families and identification of wheat nsLtp genes by EST data mining[J]. BMC Genomics,2008, 9(1):86-105.
 
  [12] CHOI Y E, LIM S, KIM H J, et al. Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes[J]. The Plant Journal,2012, 70(3):480-491.
 
  [13] TSUBOI S, OSAFUNE T, TSUGEKI R, et al. Nonspecific lipid transfer protein in castor bean cotyledon cells: Subcellular localization and a possible role in lipid metabolism[J]. Journal of Biochemistry,1992,111(4):500-508.
 
  [14] HOLLENBACH B, SCHREIBER L, HARTUNG W, et al. Cadmium leads to stimulated expression of the lipid transfer protein genes in barley: Implications for the involvement of lipid transfer proteins in wax assembly[J]. Planta,1997,203(1):9-19.
 
  [15] PARK S Y, LORD E M. Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion[J]. Plant Molecular Biology,2003,51(2):183-189.
 
  [16] YEATS T H, ROSE J K C. The biochemistry and biology of extracellular plant lipid-transfer proteins(LTPs)[J]. Protein Science,2008,17(2):191-198.
 
  [17] LEE S B, GO Y S, BAE H J,et al. Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola[J]. Plant Physiology,2009, 150(1):42-54.
 
  [18] SAROWAR S, KIM Y J, KIM K D. Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco[J]. Plant Cell Reports,2009,28(3):419-427.
 
  [19] LOON V L C, STRIEN V E A. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins[J]. Physiological and Molecular Plant Pathology,1999,55(2):85-97.
 
  [20] ZOU H W, WU Z Y, YANG Q. Gene expression analyses of ZmPti1, encoding a maize Pti-like kinase, suggest a role in stress signaling[J]. Plant Science,2006,171(1):99-105.
 
  [21] ZOU H W, WU Z Y, ZHANG X H. Over-expression of ZmPti1, a homologue to Pti1, increases salt tolerance of Arabidopsis thaliana[J]. African Journal of Biotechnology,2010, 9(5):656-662.

   论文榜(www.zglwb.com),是一个专门从事期刊推广、论文发表的网站。
本站提供如何发表论文,寻求论文发表代理,快速发表论文,发表论文格式指导等解决方案:省级论文发表/国家级论文发表/核心期刊论文发表//职称论文发表。


栏目列表
联系方式
推荐内容
 
QQ在线咨询
论文发表热线:
189-6119-6312
微信号咨询:
18961196312
期刊导航 |  论文欣赏 |  期刊验证 |  学术答疑 |  咨询辅导 |  相关知识 |  发表须知 |  关于我们 |