CN111825755A - 吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白及其编码基因与应用 - Google Patents
吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白及其编码基因与应用 Download PDFInfo
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- CN111825755A CN111825755A CN202010734390.0A CN202010734390A CN111825755A CN 111825755 A CN111825755 A CN 111825755A CN 202010734390 A CN202010734390 A CN 202010734390A CN 111825755 A CN111825755 A CN 111825755A
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Abstract
吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白及其编码基因与应用。一种氨基酸序列如SEQ ID NO.2所示的小白菜质膜内在水通道蛋白，其编码基因BraPIP2；1的核苷酸序列如SEQ ID NO.1所示，该质膜内在水通道蛋白具有灵敏地响应外界环境中新烟碱类杀虫剂(噻虫嗪、吡虫啉等)的特征，同时具有介导新烟碱类杀虫剂的跨膜传输的功能，促进新烟碱类杀虫剂在植株根和叶积累，在指导农药高效轻简使用、新型内吸性农药开发等方面具有重要的应用价值。
Description
技术领域
本发明属于生物技术领域，特别是一种来源于小白菜的质膜内在水通道蛋白及其在提高农作物新烟碱类杀虫剂传输利用效率中的应用。
技术背景
新烟碱类杀虫剂（如噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺和呋虫胺等）是与天然杀虫剂尼古丁结构相似的合成化合物，其靶向作用于昆虫中枢神经系统的烟碱型乙酰胆碱受体(nAChRs) ，因此具有较高的选择性，与传统杀虫剂相比对非靶标生物毒性较低，新烟碱类杀虫剂极性强，溶解性高，内吸作用强，用于茎叶喷雾、土壤灌根、种子处理，施药后叶片或根系通过内吸迅速传输到植株各部位，可有效防治蚜虫、潜叶蛾等害虫，现已在世界范围内广泛使用，占据了杀虫剂市场的24%及种衣剂市场的80%。。
水通道蛋白主要促使水分的双向跨膜运动, 它所介导的自由水快速被动地跨生物膜转运, 是水进出细胞的主要途径。一般而言，植物水通道蛋白都是MIP超家族的成员，不同植物的水通道蛋白分子结构同源性很高，序列非常保守。在分子结构方面，都有典型的6个跨膜结构域和5个环，并且被分别命名为A、B、C、D和Ｅ环，所有的成员在Ｂ环和Ｅ环都含有一个保守性非常高的NPA结构（Asn-Pro-Ala，NPA）。Johanson等（Johanson U. Thecomplete set of genes encoding major intrinsic proteins in Arabidopsisprovides a framework for a new nomenclature for major intrinsic proteins inplants. Plant Physiology, 2001, 126(4):1358-69.）根据氨基酸序列同源性和亚细胞定位将水通道蛋白划分为5个家族：质膜内在蛋白(PIPs)， 液泡膜内在蛋白(TIPs)，类Nodulin26膜内在蛋白(NIPs)，小的碱性膜内在蛋白(SIPs)和类GlpF膜内在蛋白(GIPs)。球蒴藓中除了有上述5类水通道蛋白以外，还具有XIPs和HIPs等2个新类别 (Bienert G P,Bienert M D, Jahn T P, et al. Solanaceae XIPs are plasma membrane aquaporinsthat facilitate the transport of many uncharged substrates. Plant Journal,2011, 66(2):306-317.) 。
除了转运水分子以外，研究发现水通道蛋白还能运输一些小分子物质如甘油、硅酸、铵、尿素、硼酸、CO2、亚砷酸盐、H2O2和氨等。一些茄科 XIPs 还能够促进一些中性小分子如甘油、尿素的运输。拟南芥中，AtNIP6;1和AtNIP7;1参与硼的转运，能调节硼在地上部与花粉中的营养分配比例Li T, Choi W G, Wallace I S, et al. Arabidopsis thalianaNIP7;1: an anther-specific boric acid transporter of the aquaporinsuperfamily regulated by an unusual tyrosine in helix 2 of the transportpore. Biochemistry, 2011, 50(31): 6633-6641.)。Terashima 等（Terashima I, OnoK. Effects of HgCl2 on CO2 dependence of leaf photosynthesis: evidenceindicating involvement of aquaporins in CO2 diffusion across the plasmamembrane. Plant Cell Physiology, 2002, 43(1): 70-78.）使用 HgCl2抑制剂证明水通道蛋白能够促进 CO2在质膜上的转运，增强植物的光合作用。酵母细胞及爪蟾卵母细胞的功能表达实验表明 ZmPIP1;5，ZmPIP1;6 能够增强拟南芥保卫细胞中 CO2的运输(HeinenR B, Bienert G P, Cohen D, et al. Expression and characterization of plasmamembrane aquaporins in stomatal complexes of Zea mays. Plant MolecularBiology, 2014, 86(3): 335-350.)。水稻OsNIP2;1，OsNIP1;1，OsNIP3;3参与了水稻对亚砷酸盐的吸收，OsNIP3;2参与了水稻对三价砷的吸收（Chen Y, Sun S K, Tang Z, et al.The Nodulin 26-like intrinsic membrane protein OsNIP3;2 is involved inarsenite uptake by lateral roots in rice. Journal of Experimental Botany,2017, 68(11):3007-3016.; Sun S K, Chen Y, Che J, et al. Decreasing arsenicaccumulation in rice by overexpressing OsNIP1;1 and, OsNIP3;1throughdisrupting arsenite radial transport in roots. New Phytologist, 2018.）。Moller等（Moller I M. Plant mitochondria and oxidative stress: electron transport,NADPH turnover, and metabolism of reactive oxygen species. Annual Review ofPlant Biology, 2001, 52(4):561-591.）研究表明AQP8 能促进 H2O2从线粒体基质中的释放。
然而现有水通道蛋白相关报道涉及多个结构差异较大的小分子物质，但较大的分子（如新烟碱类杀虫剂）尚未见相关促进转运功能的报道。
发明内容
针对上述问题，本发明目的在于提供一个新的负责新烟碱类杀虫剂跨膜转运的质膜内在水通道蛋白及其编码基因，该基因是从小白菜中分离的一个DNA分子，申请人将其命名为BraPIP2;1；本发明同时还提供了质膜内在水通道蛋白在提高植物新烟碱类杀虫剂噻虫嗪转运效率中的应用。
具体而言，上述发明是通过如下技术方案实现的：
首先，本申请提供了一种质膜内在水通道蛋白，该蛋白氨基酸序列如SEQ ID NO.2所示。该质膜内在水通道蛋白来源于小白菜，定位于质膜上。通过分子对接技术发现，新烟碱类杀虫剂分子能穿过该蛋白的空洞；相对于无添加新烟碱类杀虫剂（噻虫嗪等）而言，该质膜内在水通道蛋白在添加噻虫嗪的环境中表达丰度显著增强，具有强大地介导噻虫嗪跨膜传输的特征，且加入水通道蛋白抑制剂后，可以抑制小白菜对不同新烟碱类杀虫剂的吸收。
其次，本发明还提供了上述氨基酸序列如SEQ ID NO.2所示质膜内在水通道蛋白的编码基因BraPIP2;1，其核苷酸编码序列如SEQ ID NO.1所示。该基因的克隆方法如下：采用RNA simple Total RNA Kit 试剂盒 (Tiangen Biotech, Beijing, China) 提取小白菜根的总RNA，根据Primescript 1st Strand cDNA Synthesis Kit (Invirtrogen) 试剂盒步骤，以2g总RNA 为模板，以oligo (dT)18 作为锚定引物合成第一链cDNA，根据本研究室小白菜转录组分析结果中PIP1;1的氨基酸序列信息，设计保守区的简并引物，以上述合成的第一链cDNA为模板，通过简并全长引物扩增ORF序列，扩增PCR产物送测序，申请人将其自命名为BraPIP2;1。
第三，本发明的一个实施例提供了一种含有上述编码基因BraPIP2;1的重组表达载体，进一步，该重组表达载体为酿酒酵母表达载体，该酿酒酵母表达载体包括但不限于载体pYES2。
第四，本发明的一个实施例提供了一种含有上述编码基因BraPIP2;1的双元超表达载体，该双元超表达载体包括但不限于载体pCAMBIA2301。
第五，本发明的一个实施例提供了氨基酸序列如SEQ ID NO. 2所示的质膜内在水通道蛋白在提高植物对新烟碱类杀虫剂的吸收转运效率中的应用，所述植物包括拟南芥、蔬菜（小白菜等）、水稻，玉米，小麦，油菜，中的至少一种；所述新烟碱类杀虫剂包括噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺、呋虫胺中的一种或多种。
第六，本发明的一个实施例公开了一种促进植物对新烟碱类杀虫剂吸收转运方法：将含有如SEQ ID No .1所示核苷酸序列的双元超表达载体导入植物体内表达，以促进植物对新烟碱类杀虫剂吸收转运；该双元超表达载体包括但不限于载体pCAMBIA2301；所述植物优选蔬菜、水稻、玉米、小麦、油菜、拟南芥中的至少一种；所述新烟碱类杀虫剂优选噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺、呋虫胺中的至少一种。
本发明首次发现水通道蛋白参与了新烟碱类杀虫剂的跨膜转运，并首次发现质膜内在水通道蛋白中的BraPIP2;1基因资源并对其进行功能鉴定，本发明提供的小白菜质膜内在水通道蛋白主要定位于质膜，可介导新烟碱分子的转运，具有灵敏地响应外界环境中噻虫嗪的特征，同时具有快速地介导噻虫嗪从细胞膜外到细胞膜内的功能，促进噻虫嗪在植株体内的积累，在农药开发方面具有重要的应用价值。
附图说明
图1为 BraPIP2;1蛋白的亚细胞定位示意图；
共聚焦显微镜观察的瞬时转化48小时的烟草叶片细胞。比例尺=40 µm。
图2为水通道抑制剂对小青菜吸收不同新烟碱类杀虫剂及烟碱的影响（A:噻虫嗪；B：吡虫啉；C:啶虫脒；D:烯啶虫胺；E:呋虫胺；F:烟碱）。
图3为BraPIP2;1与不同新烟碱类杀虫剂及烟碱分子对接分析。
图4为 BraPIP2;1的表达特征及对的响应特征示意图；
其中，A为根组织BraPIP2;1对外界噻虫嗪的响应特征；B为地上组织BraPIP2;1对外界噻虫嗪的响应特征。
图5 为BraPIP2;1重组酵母菌株在噻虫嗪胁迫下的生长情况和噻虫嗪含量示意图；
其中，图A为重组酵母在不同浓度噻虫嗪胁迫下的生长；B为重组酵母菌株的噻虫嗪含量示意图。
图6为 BraPIP2;1转基因拟南芥植株在噻虫嗪胁迫下的生长情况和噻虫嗪含量示意图；
其中，A为转基因拟南芥植株在不同浓度噻虫嗪胁迫下的生长情况；B为转基因拟南芥植株在不同浓度噻虫嗪胁迫下的主根长度示意图；C为转基因拟南芥植株的噻虫嗪含量示意图；BraPIP2;1#3 和BraPIP2;1#5为超表达拟南芥株系。
具体实施方式
以下结合实例进一步说明本申请的的技术方案。
实施例1 BraPIP2;1基因全长编码区的克隆及分析
1.BraPIP2;1的ORF扩增
根据用RNA simple Total RNA Kit 试剂盒 (Tiangen Biotech, Beijing, China)操作步骤，提取小白菜根部位样品的总RNA以2g总RNA为模板，以oligo (dT)18作为锚定引物，参照Primescript 1st Strand cDNA Synthesis Kit (Invirtrogen) 试剂盒说明书合成第一链cDNA，设引物进行PCR扩增获得单一的cDNA片段，引物序列如下：上游引物序列（SEQID NO.3）ATGGCGAAGGACGTGGAAGC;下游引物序列（SEQ ID NO.4）：TTAAACGTTGGCAGCACTTCTG。
扩增体系总体积50. 0 uL，包括cDNA 20 ng、5×Prime STAR buffer 10. 0 uL、2. 5 mmol·L-1 dNTPs 4. 0 uL、10 mmol·L-1 正向引物和反向引物各2. 0uL、 PrimeSTARHS DNA Polymerase 1.25 U，用重蒸水补足至50. 0 uL。
扩增程序为: 95℃预变性5 min，98 ℃变性10 s、55℃复性15 s、72 ℃延伸60 s，共35 个循环。
PCR产物送擎科生物公司进行测序，测序结果表明，BraPIP2;1的cDNA全长序列为2550 bp，BraPIP2;1的cDNA及其编码的蛋白质氨基酸序列分别如SEQ ID No.1及SEQ IDNo.2所示。
实施例2. BraPIP2;1的亚细胞定位
参照文献 Liu et al.（Liu T, et al. Unconventionally secreted effectors oftwo filamentous pathogens target plant salicylate biosynthesis. Nat Commun 5,4686. 2014）将 BraPIP2;1构建到亚细胞定位载体pBinGFP4上，通过冻融法转化到农杆菌菌株GV3101(江苏省农业科学院实验室保存)中。瞬时转化烟草，48h-60h 后在荧光显微镜下观察荧光。发现，绿色荧光信号主要分于细胞膜或核膜上(见图1)，说明BraPIP2;1主要定位于与跨膜运输有关在细胞膜和核膜上。
实施例3抑制水通道蛋白活性可降低蔬菜对新烟碱类农药的吸收累积量。
参照论文（Wenfeng W , Wan Q , Li Y , et al. Uptake, translocation andsubcellular distribution of pesticides in Chinese cabbage (Brassica rapa var.chinensis)[J]. Ecotoxicology and Environmental Safety, 2019.）在已添加2mg/L烟碱类化合物（噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺和呋虫胺、烟碱）的等体积霍格兰德营养液中添加不同浓度的水通道抑制剂氯化汞和甘油，48h后分别测定小白菜植株（三叶一心）中的烟碱类化合物的浓度。每个处理五个重复。
为确定水通道蛋白是否能转运新烟碱类杀虫剂分子及烟碱，在已添加2mg/L不同新烟碱类杀虫剂（噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺和呋虫胺）及烟碱的等体积营养液中添加不同浓度的水通道抑制剂氯化汞（HgCl2）和甘油（Glycerol），分别测定植株中的烟碱化合物浓度。
检测结果如图2所示，添加水通道抑制剂后，新烟碱在植株里的含量都极显著地降低，而烟碱在植株中的含量没有发生显著性变化。由此可知水通道抑制剂能显著抑制上海青对新烟碱类杀虫剂的吸收，而不能抑制烟碱的吸收。说明水通道蛋白参与了新烟碱类杀虫剂的跨膜转运。
实施例4 BraPIP2;1蛋白与不同新烟碱及烟碱的分子对接分析
将BraPIP2;1氨基酸序列通过同源建模的方法获得BraPIP2;1的3D结构，通过分子对接技术，以验证新烟碱类杀虫剂分子（噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺和呋虫胺）和烟碱分子能否穿过BaPIP2;1晶体结构中间的孔道，对接范围包括整个蛋白构象，因此可以通过生成的众多构象中筛选新烟碱类杀虫剂分子和烟碱分子进入蛋白中央孔径内部的对接结果，以此分析类杀虫剂分子能否穿过蛋白孔洞。
对接所用软件为AutoDockTools-1.5.6，设置对接盒子大小为100 Å*126 Å*100Å, 采用半柔性对接，算法采用拉马克遗传算法，对接生成结果200次。对接结果见图3：新烟碱类杀虫剂分子（噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺和呋虫胺）结合在蛋白的通道中央，证明噻虫嗪等新烟碱类杀虫剂分子在进入通道时不会产生空间位阻。而烟碱分子不能结合在蛋白的通道中央，证明烟碱分子在进入通道时会产生空间位阻。
以上结果说明BraPIP2;1蛋白能转运新烟碱类杀虫剂，而不能转运烟碱。
实施例5 BraPIP2;1基因对环境噻虫嗪胁迫的响应
将生长情况一致的小白菜上海青幼苗置于含有10mg/L噻虫嗪的Hoagland's培养液（Hoagland's营养液购自北京酷来搏科技有限公司），在Hoagland's培养液中处理6和24小时（处理组）。
同时不含有噻虫嗪的Hoagland's培养液为对照组；
分别提取两组处理根和地上组织的RNA，反转录获得第一链cDNA作为模板，根据BraPIP2;1的cDNA序列设计特异性表达引物，以小青菜Tublin为内参，通过定量RT-PCR检测基因转录水平的表达情况。
定量PCR引物设计：
BraPIP2;1-F(SEQ ID NO .5）: TCACTGGAACTGGAATCAACCC；
BraPIP2;1-R(SEQ ID NO .6）: CCTGAAGCCCTCAAAACGAAC；
Tublin-F(SEQ ID NO .7）: ACTGGGTGTTTTGGGTTGGG；
Tublin-R(SEQ ID NO .8）: TGAAGGGGATTGCTCTGATGAC；
定量仪器为7500 Real Time PCR System (Applied Biosystem)，参照试剂盒2×TSINGKE Master qPCR Mix (擎科生物公司)的说明书配置qPCRT体系；PCR程序为：预变性95℃ 10 min；95℃ 15s，60℃ 20s，40个循环。
qRT-PCR结果表明，BraPIP2;1在地上和地下的都表达，与对照相比，在添加噻虫嗪的条件下BraPIP2;1在处理6小时和24小时后，根和地上部位的的表达丰度极显著增强（p<0.001）(如图4所示)，说明噻虫嗪的传输利用与BraPIP2;1通道蛋白具有相关性。
实施例6. 超表达BraPIP2;1基因酿酒酵母可提高对噻虫嗪的胁迫耐受。
将通过实施例1克隆得到的BraPIP2;1基因构建到酿酒酵母过表达载体pYES2（购自Clotech公司）上参照文献（贺琳， 蒋丽丽， 王玉成，柽柳硫氧还蛋白过氧化物酶(ThPrx1)基因转入酵母的抗逆能力分析，东北林业大学学报，2011, 第39卷. 第4期, 101-104），构建的重组质粒命名为 pYES2-BraPIP2;1。将pYES2-BraPIP2;1及空载体 pYES2 用乙酸锂沉淀法转化酿酒酵母 INVSc1 ( Saccharomyces cerevisiae) ，重组酵母分别命名为 INVSc1 ( pYES2-BraPIP2;1) 和 INVSc1( pYES2)。
重组菌株的诱导：挑取对照酵母 INvscl( pYES2) 和重组酵母 INvscl( pYES2-BraPIP2;1) 单菌落，分别接种于 SC-U 液体培养基( 加入终浓度 2% 葡萄糖) 中，30℃摇床振荡培养 24 h，测其 OD600值，计算所需菌液量，使 10 m L 诱导培养基 ( SC-U + 2%半乳糖) 中菌体 OD600为 0.4，30 ℃诱导表达 24 h; 再次测其 OD600值，计算所需的菌液量。
重组菌株的噻虫嗪胁迫生长实验：参照文献“贺琳， 蒋丽丽， 王玉成，柽柳硫氧还蛋白过氧化物酶(ThPrx1)基因转入酵母的抗逆能力分析，东北林业大学学报，2011， 第39卷. 第4期, 101-104”中公开的方法，测定诱导后的菌液 OD600值，计算所需的菌液量，使200 μL 体积中菌体 OD600为 2，8 500 r/min，离心 1 min，弃上清。
噻虫嗪胁迫处理: 将菌体分别重悬于 200 μL 0, 10, 100, 400, 800, 1600 mg/ L 的噻虫水嗪溶液中，30℃ 胁迫 72 h。将菌液依次稀释 10、100、1 000、10 000 、100000倍后取 3 μL 涂在 SC-U( 加入终浓度为 2% 的葡萄糖) 的固体培养基上; 30 ℃ 培养 48 h。结果如图5A所示：转基因菌株（pYES2-BraPIP2;1）在不添加噻虫嗪情况下，生长与野生菌株（pYES2）一致，添加噻虫嗪的情况下，生长明显弱于野生将菌株。
重组菌株的噻虫嗪含量实验：测定诱导后的菌液 OD600值，计算所需的菌液量，使500ml体积中菌体 OD600为 0.1，8 500 r/min，离心 1 min，弃上清。将菌体重悬于500ml分别含10和100 mg/ L 噻虫嗪的SC-U 液体培养基( 加入终浓度 2% 葡萄糖)中，30 ℃ 胁迫 6 h，测定OD600值。6000 rpm，10min离心后，菌体用超纯水洗三次后，菌体用玻璃珠结合超声的方法破壁后提取噻虫嗪测定量。在10 mg/ L噻虫嗪胁迫下，重组菌株的噻虫嗪含量为0.0071 ng/OD极显著高于对照菌株（0.0038 ng/OD）, 在100 mg/ L噻虫嗪胁迫下，重组菌株（pYES2-BraPIP2;1）的噻虫嗪含量为0.028 ng/OD极显著高于对照菌株（pYES2，0.011ng/OD）（见图5B）。
实施例7 .BraPIP2;1基因在拟南芥超表达可提高拟南芥对噻虫嗪的吸收和累积量
将通过实施例1克隆得到的BraPIP2;1基因构建到植物双元超表达载体pCAMBIA2301（购自Clotech公司）上，通过根癌农杆菌介导方法（Valvekens, D., van Montagu, M.,and Lijsebettens, M . V. (1988). Agrobacterium tumefaciens-mediatedtransformation of Arabidopsis thaliana root explantsby using kanamycinselection. Proc. Natl. Acad. Sci. USA 85, 5536-5540.）将构建的超表达载体侵染野生型拟南芥Col-0（购自美国ATCC）的花絮，通过筛选（在含50μg/mL卡那霉素的1/2MS平板上筛选抗性纯系苗），鉴定获得BraPIP2;1超表达拟南芥纯系材料BraPIP2;1#3及BraPIP2;1#5。
分别在1/2MS培养基萌发前面转基因实验得到的BraPIP2;1#3及BraPIP2;1#5的纯系种子及野生型Col-0材料（WT），再转入分别含10、50mg/L的噻虫嗪的1/2MS培养基(青岛海博生物技术有限公司， 货号 HB8469-12 PH=5.6)中，20天后观察幼苗生长情况并测定主根长度,结果分别如图6A、B所示，转基因株系的主根长短低于野生型。
此外将萌发的大小一致的苗移栽至含10mg/L噻虫嗪的土壤中，培养20天后分别收获转基因拟南芥及对照的根部和地上部样品，用去离子水洗净，称量鲜重，参照文献（Wenfeng W , Wan Q , Li Y , et al. Uptake, translocation and subcellulardistribution of pesticides in Chinese cabbage (Brassica rapa var. chinensis)[J]. Ecotoxicology and Environmental Safety, 2019.）测定和计算各样品的噻虫嗪含量，结果如图6C所示：转基因植株的根和地上部分的噻虫嗪含量高于野生株。
以上实施例说明，水通道蛋白BraPIP2;1基因资源具有灵敏地响应外界环境中新烟碱类杀虫剂的特征，同时具有快速地介导新烟碱类农药吸收和传输的功能，促进新烟碱类农药在植株体内的积累，在改良（农）作物利用农药方面及农药开发方面具有重要的应用价值，为进一步通过改造农药结构开发内吸性农药来提高农药利用率奠定了基础。
序列表
<110> 江苏省农业科学院
<120> 吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白及其编码基因与应用
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atggcgaagg acgtggaagc cgtttccgga gaagggtttc agacaagaga ctatcaagat 60
ccaccgccag cgcctctgtt tgacccggcg gagctgacca agtggtcttt ctacagagca 120
gtcatcgctg agttcgtagc tactctcctc ttcctgtaca tcaccgtttt gacagtcatc 180
ggttacaaga ttcagaccga tagtactgcc ggcggcgttg actgcggtgg agttggtatc 240
ctaggtatcg cttgggcttt tggtggcatg atcttcatcc ttgtctactg caccgccggt 300
atctctggtg gtcacattaa cccggcagtg acatttgggc tactcttagc gcgtaaagtg 360
tccttggtaa gagccatttt gtacatggta gctcagtgtt tgggtgcgat atgcggagtt 420
gggttcgtca aagccttcca aagctcttac tacgtccgtt acggtggtgg agcaaactct 480
ctagccgacg gctacagcac aggaaccgga ctagccgcag agatcattgg tactttcgtt 540
cttgtctaca ccgtcttctc cgccactgac cctaaacgta gtgccagaga ctcccacgtc 600
ccagtgttgg cgccacttcc cattggattt gcggtgttca tggtgcactt ggctaccatt 660
cccatcactg gaactggaat caacccggca aggagtttcg gagcagccgt tatcttcaac 720
gagagcaagc catgggatga ccactggata ttttgggtgg gaccattcgt tggagctgcg 780
atagctgcat tctaccacca gttcgttttg agggcttcag gttccaagtc tctcggatct 840
ttcagaagtg ctgccaacgt ttaa 864
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<211> 287
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Met Ala Lys Asp Val Glu Ala Val Ser Gly Glu Gly Phe Gln Thr Arg
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Asp Tyr Gln Asp Pro Pro Pro Ala Pro Leu Phe Asp Pro Ala Glu Leu
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Thr Lys Trp Ser Phe Tyr Arg Ala Val Ile Ala Glu Phe Val Ala Thr
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Leu Leu Phe Leu Tyr Ile Thr Val Leu Thr Val Ile Gly Tyr Lys Ile
50 55 60
Gln Thr Asp Ser Thr Ala Gly Gly Val Asp Cys Gly Gly Val Gly Ile
65 70 75 80
Leu Gly Ile Ala Trp Ala Phe Gly Gly Met Ile Phe Ile Leu Val Tyr
85 90 95
Cys Thr Ala Gly Ile Ser Gly Gly His Ile Asn Pro Ala Val Thr Phe
100 105 110
Gly Leu Leu Leu Ala Arg Lys Val Ser Leu Val Arg Ala Ile Leu Tyr
115 120 125
Met Val Ala Gln Cys Leu Gly Ala Ile Cys Gly Val Gly Phe Val Lys
130 135 140
Ala Phe Gln Ser Ser Tyr Tyr Val Arg Tyr Gly Gly Gly Ala Asn Ser
145 150 155 160
Leu Ala Asp Gly Tyr Ser Thr Gly Thr Gly Leu Ala Ala Glu Ile Ile
165 170 175
Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser Ala Thr Asp Pro Lys
180 185 190
Arg Ser Ala Arg Asp Ser His Val Pro Val Leu Ala Pro Leu Pro Ile
195 200 205
Gly Phe Ala Val Phe Met Val His Leu Ala Thr Ile Pro Ile Thr Gly
210 215 220
Thr Gly Ile Asn Pro Ala Arg Ser Phe Gly Ala Ala Val Ile Phe Asn
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Glu Ser Lys Pro Trp Asp Asp His Trp Ile Phe Trp Val Gly Pro Phe
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Val Gly Ala Ala Ile Ala Ala Phe Tyr His Gln Phe Val Leu Arg Ala
260 265 270
Ser Gly Ser Lys Ser Leu Gly Ser Phe Arg Ser Ala Ala Asn Val
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actgggtgtt ttgggttggg 20
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Claims (10)
1. 一种吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白，其氨基酸序列如SEQ ID No.2所示。
2.编码如权利要求1所述吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白的基因序列，其核苷酸序列如SEQ ID No .1所示。
3.一种含有如SEQ ID No .1所示核苷酸序列的重组表达载体。
4.一种含有如SEQ ID No .1所示核苷酸序列的双元超表达载体。
5.如权利要求1所述吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白在促进植物对新烟碱类杀虫剂吸收转运中的应用。
6.如权利要求5所述的应用，其特征在于，所述植物包括拟南芥、蔬菜、水稻、玉米、小麦、油菜中的至少一种。
7.如权利要求5所述的应用，其特征在于，所述新烟碱类杀虫剂包括噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺、呋虫胺中的至少一种。
8.一种促进植物对新烟碱类杀虫剂吸收转运方法，其特征在于，具体步骤如下：将含有如SEQ ID No .1所示核苷酸序列的双元超表达载体导入植物体内表达，以促进植物对新烟碱类杀虫剂吸收转运。
9.如权利要求8所述促进植物对新烟碱类杀虫剂吸收转运方法，其特征在于，所述植物包括蔬菜、水稻、玉米、小麦、油菜、拟南芥中的至少一种。
10.如权利要求8所述促进植物对新烟碱类杀虫剂吸收转运方法，其特征在于，所述新烟碱类杀虫剂包括噻虫嗪、吡虫啉、啶虫脒、烯啶虫胺、呋虫胺中的至少一种。
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