Isolation of Two Genes Encoding Nonspecific Lipid Transfer Protein and Their Expression Profiles in Ipomoea batatas(英文稿)
-
摘要: 非特异脂质转移蛋白（nsLTP）是植物界普遍存在的一类涉及多种胁迫反应的可溶性蛋白。为了阐明甘薯中非特异性脂质转移蛋白基因IbLTP1和IbLTP2在盐胁迫反应中的功能，本研究运用PCR技术，对IbLTP1和IbLTP2基因进行了克隆，并通过生物信息学方法分析了序列结构、蛋白质保守结构域和系统进化关系；利用qRT-PCR方法检测了这两个基因在不同组织中的表达模式以及盐胁迫条件下的表达差异；将IbLTP1和IbLTP2基因克隆到大肠杆菌的原核表达载体pET32a中，对重组菌BL21（pET32a-LTP）的耐盐性进行分析。序列分析表明：IbLTP1和IbLTP2编码区均不含内含子并都具有等位基因。IbLTP1和IbLTP2基因的蛋白质序列分别包括114和94个氨基酸残基并且不含色氨酸残基，蛋白序列N端含有信号肽序列。保守结构域和系统进化分析结果表明：IbLTP1和IbLTP2均含有nsLTP蛋白的保守结构域，IbLTP1属于Type Ⅰ而IbLTP2属于Type Ⅱ。实时荧光定量PCR分析表明：IbLTP1在幼叶中表达量最高，根中表达量最低；而IbLTP2在茎中表达量最高，成熟叶中表达量最低。在NaCl胁迫条件下，IbLTP1和IbLTP2表达量在根中基本无变化而在茎和叶中上调。大肠杆菌BL21（DE3）中异源表达IbLTP1和IbLTP2基因能够提高转基因菌株对NaCl的耐受性。因此，本研究推测IbLTP1和IbLTP2基因可能在甘薯盐胁迫反应中发挥了作用。Abstract: Nonspecific lipid transfer proteins (nsLTPs) are widely distributed in the plant kingdom and are involved in various stress responses. To clarify the function of nsLTP genes, IbLTP1 and IbLTP2 were cloned by PCR technology, and the sequence structures, conserved domains, and evolutionary relationships were analyzed.Sequences of cDNAs and genomic genes showed that neither gene had introns, but both had several homologous isoforms. IbLTP1 and IbLTP2 encode proteins of 114 and 94 amino acid residues respectively, without any Trp. These proteins contain a signal peptide at the N-terminal and have conserved domains of nsLTP1 and nsLTP2, respectively. The expression patterns and expression differences of IbLTP1 and IbLTP2 in different tissues and under stress were determined by real-time RT-PCR. Results showed that IbLTP1 and IbLTP2 had higher relative expression levels in young leaves and stems, respectively, and were highly induced under sodium chloride (NaCl) stress. The coding sequences of IbLTP1 and IbLTP2 were cloned into expression vector pET32a and expressed in Escherichia coli BL21 (DE3), respectively. The maximal OD600 values of strains harboring pET32a-IbLTP1 and pET32a-IbLTP2 were higher than those of the pET32a transformed strain under NaCl stress.
-
-
[1] Sossountzov L, Ruiz-Avila L, Vignols F, Jolliot A, Arondel V, Tchang F, Grosbois M, Guerbette F, Miginiac E, Delseny M. Spatial and temporal expression of a maize lipid transfer protein gene[J]. Plant Cell, 1991, 3(9): 923-933.
[2] Arondel V, Kader JC. Lipid transfer in plants[J]. Experientia, 1990, 46(6): 579-585.
[3] Castagnaro A, García-Olmedo F. A fatty-acid-binding protein from wheat kernels[J]. Febs Lett, 1994, 349(1): 117-119.
[4] Carvalho ADO, Gomes VM. Role of plant lipid transfer proteins in plant cell physiology-A concise review[J]. Peptides, 2007, 28(5): 1144-1153.
[5] Wei KF, Zhong XJ. Non-specific lipid transfer proteins in maize[J]. BMC Plant Biol, 2014, 14(1): 1-18.
[6] Boutrot F, Chantret N, Gautier MF. 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]. J Comp Neurol, 2007, 140(2): 155-173.
[7] Dong HS, Lee JY, Hwang KY, Kim KK, Suh SW. High-resolution crystal structure of the non-specific lipid-transfer protein from maize seedlings[J]. Structure, 1995, 3(3): 189-199.
[8] Samuel D, Liu YJ, Cheng CS, Lyu PC. Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa)[J]. J Biol Chem, 2002, 277(38): 35267-35273.
[9] Chen Y, Ma JJ, Zhang X, Yang YT, Zhou DG, Yu Q, Que YX, Xu LP, Guo JL. A novel non-specific lipid transfer protein gene from gugarcane (NsLTPs ), obviously responded to abiotic stresses and signaling molecules of SA and MeJA[J]. Sugar Tech, 2016: 1-9.
[10] Qin XY, Liu Y, Mao SJ, Li TB, Wu HK, Chu CC, Wang YP. Genetic transformation of lipid transfer protein encoding gene in Phalaenopsis amabilis, to enhance cold resistance[J]. Euphytica, 2011, 177(1): 33-43.
[11] Jang CS, Lee HJ, Chang SJ, Yong WS. Expression and promoter analysis of the TaLTP1, gene induced by drought and salt stress in wheat (Triticum aestivum L.)[J]. Plant Sci, 2004, 167(5): 995-1001.
[12] Guan MX, Chai RH, Kong X, Liu XM. Isolation and characterization of a lipid transfer protein gene (BplLTP1) from Betula platyphylla[J]. Plant Mol Biol Rep, 2013, 31(4): 991-1001.
[13] George S, Parida A. Characterization of an oxidative stress inducible nonspecific lipid transfer protein coding cDNA and its promoter from drought tolerant plant Prosopis juliflora[J]. Plant Mol Biol Rep, 2010, 28(1): 32-40.
[14] Manjula S, Murali M, Shivamurthy GR, Amruthesh KN. Non-specific lipid transfer proteins (ns-LTPs) from maize induce resistance in pearl millet against downy mildew disease[J]. Phytoparasitica, 2014, 43(4): 437-447.
[15] Lee SB, Go YS, Bae HJ, Park JH, Cho SH, Cho HJ, Lee DS, Park OK, Hwang I, Suh MC. Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglo-bules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola[J]. Plant Physiol, 2009, 150(1): 42-54.
[16] Huang MD, Chen TL, Huang AH. Abundant Type Ⅲ lipid transfer proteins in Arabidopsis tapetum are secreted to the locule and become a constituent of the pollen exine[J]. Plant Physiol, 2013, 163(3): 1218-1229.
[17] Martín-Pedraza L, González M, Gómez F, Blanca-López N, Garrido-Arandia M, Rodríguez R, Torres MJ, Blanca M, Villalba M, Mayorga C. Two non-specific lipid transfer proteins (nsLTP) from tomato seeds are associated to severe symptoms of tomato-allergic patients[J]. Mol Nutr Food Res, 2016, 60(5): 1172-1182.
[18] Xie FL, Burklew CE, Yang YF, Liu M, Xiao P, Zhang BH, Qiu DY. De novo sequencing and a comprehensive analysis of purple sweet potato (Ipomoea batatas L.) tran-scriptome[J]. Planta, 2012, 236(1): 101-113.
[19] Wang ZY, Fang BP, Chen JY, Zhang XJ, Luo ZX, Huang LF, Chen XL, Li YJ. De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas)[J]. Bmc Genomics, 2010, 11(53): 1-14.
[20] Firon N, LaBonte D, Villordon A, Kfir Y, Solis J, Lapis E, Perlman T, Doron-Faigenboim A, Hetzroni A, Althan L, Nadir L. Transcriptional profiling of sweetpotato (Ipomoea batatas) roots indicates down-regulation of lignin biosynthesis and up-regulation of starch biosynthesis at an early stage of storage root formation[J]. BMC Genomics, 2013, 14(1): 1-25.
[21] Tao X, Gu YH, Wang HY, Zheng W, Li X, Zhao CW, Zhang YZ. Digital gene expression analysis based on integrated de novo transcriptome assembly of sweet potato (Ipomoea batatas (L.) Lam.)[J]. Plos One, 2012, 7(4): 1-14.
[22] Tao X, Gu YH, Jiang YS, Zhang YZ, Wang HY. Transcriptome analysis to identify putative floral-specific genes and flowering regulatory-related genes of sweet potato[J]. Biosci Biotech Bioch, 2013, 77(11): 2169-2174.
[23] Chen J, Jiang YS, Tao X, Tan XM, Zhang YZ. Cloning and expression profile of betaine aldehyde dehydrogenase gene of Ipomoea batatas in response to salt stress[J]. Russ J Plant Physl, 2014, 61(4): 509-516.
[24] Park SC, Kim YH, Jeong JC, Kim CY, Lee HS, Bang JW, Kwak SS. Sweetpotato late embryogenesis abundant 14(IbLEA14) gene influences lignification and increases osmotic-and salt stress-tolerance of transgenic calli[J]. Planta, 2011, 233(3): 621-634.
[25] Qin H, Zhou S, Zhang YZ. Characterization and expression analysis of starch branching enzymes in sweet potato[J]. Hypertension, 2013, 12(9): 1530-1539.
[26] Yan L, Gu YH, Tao X, Lai XJ, Zhang YZ, Tan XM, Wang HY. Scanning of transposable elements and analyzing expression of transposase genes of sweet potato (Ipomoea batatas)[J]. Plos One, 2014, 9(3): 1-18.
[27] Seo SG, Kim JS, Yang YS, Jun BK, Kang SW, Lee GP, Kim W, Kim JB, Lee HU, Kim SH. Cloning and characterization of the new multiple stress responsible geneⅠ (MuSI) from sweet potato[J]. Genes Genom, 2010, 32(6): 544-552.
[28] Pyee J, Yu H, Kolattukudy P. Identification of a lipid transfer protein as the major protein in the surface wax of broccoli (Brassica oleracea) leaves[J]. Arch Biochem Biophys, 1994, 311(2): 460-468.
[29] Thoma S, Hecht U, Kippers A, Botella J, De Vries S, Somerville C. Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis[J]. Plant Physiol, 1994, 105(1): 35-45.
计量
- 文章访问数: 1304
- HTML全文浏览量: 0
- PDF下载量: 877
下载:
