转毕式海蓬子水通道蛋白基因SbPIP1烟草的抗旱生理机制分析

Analysis of Physiological Mechanism of Drought Tolerance in SbPIP1 Transgenic Tobacco

为了研究毕氏海蓬子中水通道蛋白基因的功能,利用RACE技术克隆了毕氏海蓬子经盐诱导后差异表达的水通道蛋白基因SbPIP1,并利用生物学软件对其编码的氨基酸序列进行了分析。进一步构建SbPIP1基因的植物高效表达载体pcAMBIA2301-SbPIP1,利用农杆菌介导法转化烟草NC89,通过Kan抗性、PCR及半定量RT-PCR筛选鉴定阳性转基因苗;对转基因烟草进行抗旱性分析,同时测定转基因烟草叶片原生质体在低渗溶液中破裂所需时间,以及在不同浓度PEG6000处理后转基因烟草叶片的相对含水量(RWC)、离子外渗率(IL)、丙二醛(MDA)含量和脯氨酸含量,研究毕式海蓬子SbPIP1基因的功能,分析转基因烟草的抗旱生理机制。结果表明,从毕氏海蓬子克隆的SbPIP1基因(Gen Bank登录号:DQ451602)c DNA全长1 268 bp,开放阅读框长度为858 bp,共编码285个氨基酸。生物信息学分析显示,SbPIP1编码的蛋白包含6个跨膜区、2个NPA(Asn-Pro-Ala)单元和2个典型的质膜信号肽序列。通过Kan抗性、PCR及半定量RT-PCR鉴定筛选,最终获得10个株系转基因烟草苗。转基因植株的抗旱性与对照相比明显提高。转基因烟草叶片原生质体在低渗溶液中的破裂时间明显短于对照烟草。在不同浓度PEG6000处理下,随着处理浓度的提高,转基因烟草和对照烟草叶片RWC均呈现逐渐下降趋势;但在相同浓度处理下,转基因烟草植株叶片的RWC与对照植株相比,下降幅度较小,具有较强的抗脱水能力。转基因烟草和对照烟草叶片MDA和IL随着PEG处理浓度的提高均呈现逐渐上升趋势,但转基因烟草MDA和IL增加幅度明显小于对照植株。转基因烟草和对照烟草叶片中脯氨酸含量随着PEG处理浓度的提高也呈现逐渐上升趋势,但与对照相比,转基因烟草植株叶片中脯氨酸含量增加的更快。

In order to study the function of aquaporin genes from Salicornia bigeloii,a new plasma membrane aquaporin gene SbPIP1 was cloned from S. bigeloii based on the rapid amplification of c DNA ends（ RACE） technique and the amino acid sequence encoded by it was analyzed using bioinformatics software. The SbPIP1 gene driven by constitutive promoter Ca MV 35 S was transformed into tobacco NC89 with Agrobaeterium EHA105 containing the plant expression vector pc AMBIA2301-SbPIP1. The transgenic tobacco plants were screened by Kan resistance,PCR and semi-quantitative RT-PCR. Drought tolerance of transgenic tobacco plants was detected,and relative water content（ RWC）,malondialdehyde（ MDA）,Ion leakage（ IL） and proline content of leaves of transgenic tobacco treated with different concentrations of PEG6000 were measured. The results showed that the full-length c DNA of SbPIP1 gene（ Gen Bank Accession No. DQ451602） consists of 1 268 bp and contains a 858 bp open reading frame（ ORF） encoding 285 amino acid. Bioinformatics analysis showed that SbPIP1 protein contained a typical structure with six membrane-spanning domains,two highly conserved Asn-Pro-Ala（ NPA） motifs and two topical signal consensus sequence of plasma membrane. 10 lines of positive transgenic tobacco were finally identified by Kan resistance,PCR and semi-quantitative RT-PCR. Transgenic plants which overexpressed SbPIP1 displayed improved tolerance against drought stress when compared to WT tobacco plants. Burst time of protoplasts from transgenic tobacco plants in hypotonic solution was significantly shorter than the control tobacco. Under PEG6000 treatments with different concentrations,the RWC in transgenic and control plants decreased gradually with the increase of the treatment concentration,however,at the same treatment concentration,the RWC of transgenic tobacco leaves were higher than that of control plants. With the increase of the treatment concentration of PEG6000,MDA and IL of the transgenic and the control plants increased gradually,but those of the transgenic plants increased more slowly than those of control plants. With the increase of the PEG concentration,the proline content of transgenic and control plants also gradually increased,but proline content of the transgenic plants were higher than those of control plants.