【作者】 魏清江；

【导师】 彭抒昂；

【作者基本信息】 华中农业大学 ， 果树学， 2013， 博士

【摘要】 柑橘是一种重要的经济作物,但对盐胁迫较为敏感,尤其对含氯的盐分敏感。柑橘在盐胁迫下会积累大量的Na+和Cl-,导致植株光合下降、生长变缓并最终降低产量和果实品质。前人对柑橘耐盐性的研究主要集中在砧木上,而对不同柑橘品种间耐盐性的研究较少。此外,尽管Cl-转运在柑橘耐盐中起重要作用,但参与Cl-转运相关基因的研究还很缺乏。本研究比较了四个常见柑橘品种对NaCl胁迫的生理响应,分析了四个C1-转运相关的基因在这些盐胁迫品种叶片中的表达。此外,本研究在枳中克隆了其中的两个基因：PtrCLC和PtrCCC,并分析了基因的表达模式。为进一步研究基因在Cl-平衡中的作用,我们还将PtrCLC和PtrCCC分别在模式植物中进行了过表达,并分析了转基因植株和对照植株在盐胁迫下的表现。本研究的主要结果如下：1.比较了枳砧甜橙(纽荷尔和伦晚)和宽皮柑橘(鄂柑一号和国庆一号)对不同浓度NaCl (0、30、60和90mmol/L)胁迫的生理响应。结果表明：盐胁迫导致植株的干重、叶面积和净光合速率下降,但宽皮柑橘的下降幅度较小。与甜橙相比,宽皮柑橘叶片中积累的Na+和Cl-较低,而两种离子在植株根中的积累量较高。盐胁迫也导致品种间主要矿质元素含量的变化,而宽皮柑橘叶片中Ca和Mg的下降幅度低于甜橙。2.利用同源克隆法从枳中分离了一个CLC基因：PtrCLC。该序列包含2367bP的ORF,编码一个含789氨基酸的多肽。PtrCLC蛋白与其他物种CLC序列的一致性较高,该蛋白还含有10个跨膜区和两个CBS结构域。Real-time PCR分析表明PtrCLC在枳叶片和根中都有表达,同时该基因的表达在ABA、4℃和NaCl处理后上调。此外,在拟南芥AtCLCc突变体clcc中过表达35S::PtrCLC后发现,转基因植株的种子萌芽率在盐胁迫下得到提高。此外,在200mmol/L NaCl处理下,转基因拟南芥鲜重、电解质渗透和叶绿素含量的下降都小于突变体和野生型植株,并且转基因植株根部和地上部的C1-含量均低于突变体和野生型植株。3.为获得柑橘CLC基因家族的所有成员序列,我们用PtrCLC序列检索甜橙基因组数据库,共得到6个CLC基因序列。在枳中克隆验证了另外5个CLC基因。这6个CLC蛋白(PtrCLC1-6)长度在748-798个氨基酸之间,但成员之间序列差异较大。进化分析表明PtrCLCs可分为两个亚类,而PtrCLC4和PtrCLC6与细菌CLCs的关系相对较近。在大肠杆菌EcCLCA中,有三个与Cl-选择性吸收相关的位点：丝氨酸S107、谷氨酸E148和谷氨酸E203。序列比对发现,PtrCLC1在S107相对应位置为脯氨酸(P177), PtrCLC3在E148相对应位置为丙氨酸(A213),PtrCLC4和PtrCLC6在E203相对应位置分别为苏氨酸(T260)和丝氨酸(S295)。Real-timePCR分析表明PtrCLCs在枳叶片中偏好性表达,尤其是PtrCLC6.此外,我们分析了PtrCLCs对缺氮、NaC1和ABA处理的响应情况。缺氮后,PtrCLCs的表达都下调,而PtrCLCl在补充2mmol/L NO3-可后恢复表达。相反,NaCl处理导致PtrCLCs表达量增加,但PtrCLC2和PtrCLC4增加更明显,同时这两者也能够被ABA诱导上调表达。4.利用同源克隆法从枳中分离了一个CCC基因,PtrCCC。该序列长3438bp包含2943bP的ORF,编码一个含980氨基酸的多肽。PtrCCC蛋白含有11个跨膜区域。进化分析表明,PtrCCC与动物的KCC类转运蛋白进化关系较近。Real-timePCR分析表明PtrCCC在枳叶尖和根尖等幼嫩组织中的表达量高于成熟组织。此外,其表达能被KCl胁迫诱导上调。我们进一步将PtrCCC转入野生型烟草中进行基因的功能验证。在NaCl、KCl和NaCl+KCl胁迫下,转基因植株的生长表现都好于野生型植株。同时,在这三种盐胁迫尤其是在KCl胁迫下,转基因植株的根干重和地上部干重的积累都高于野生型植株。更多还原

【Abstract】 Citrus, one of the most important fruit crops in the world, is hypersensitive to salt stress, particularly to the salt contained Cl". The citrus plants could accumulate high concentration of Na+and Cl-under salinity, which inhibit leaf photosynthesis, retard plant growth, and finally reduce yield and fruit quality. Previous studies on salt stress in citrus mainly focused on the rootstock, and fewer studies have been done on cultivars grafted on same rootstock. In addition, although Cl-transport plays an important role in citrus response to salinity, studies on the genes involved in Cl-transport are relative lacking. In the present study, we compared the difference in physiological responses of four common citrus cultivars under various NaCl concentrations and analyzed expression of four genes involved in Cl" transport in these salt-treated plants. In addition, two of the four genes, PtrCLC and PtrCCC, were isolated from trifoliate orange. Furthermore, the PtrCLC and PtrCCC were over-expressed in model plants to identify their possible functions in Cl" homeostasis. Responses of the transgenic and non-transgenic plants to salinity were then compared. The main results of this study are as follow:1. To compare the different responses to salt stress of citrus, four common cultivars belonging to sweet oranges (Newhall and Lane late) and loose-skin mandarins (E-gan No.l and Guoqing No.l) were grafted on trifoliate orange and exposed to0,30,60or90mmol/L NaCl for two months. Results showed that the NaCl stress induced less reduction in dry weight, leaf area, and photosynthetic rate in loose-skin mandarins than that in sweet oranges. It was further found that, as compared with sweet oranges, the loose-skin mandarins accumulated less Na+and Cl" in leaves while more these ions in their roots. Meanwhile, the changes in main nutrient contents varied among the salt-treated cultivars, and loose-skin mandarins showed less decreases in leaf Mg and Ca than those in sweet oranges.2. A putative CLC encoding gene, PtrCLC, was isolated from trifoliate orange using homologous cloning. The PtrCLC contained an ORF of2,367bp, encoding a protein of788amino acids. The deduced amino acids of PtrCLC shared high identity with other CLC-like sequences, which also contained ten transmembrane regions (TMs) and two cystathionine beta-synthase (CBS) domains. Real-time PCR analysis revealed that the PtrCLC gene expressed in the leaves and roots of trifoliate orange was up-regulated by ABA,4℃and NaCl. Transformation of Arabidopsis AtCLCc mutant clcc with35S:: PtrCLC improved the seed germination of transgenic plants under salinity. In addition, the reduction in fresh weight, electrolyte leakage and chlorophyll content was lower in the transgenic seedlings than that in mutant or wild-type at200mmol/L NaCl treatment. This was further supported by the observation that the total Cl" accumulated in the roots and shoots was lower in transgenic plants than that in mutant or wild-type.3. To obtain all CLC sequences in citrus, the PtrCLC were subjected to the orange genome database, which resulted in six putative CLC sequences. The other five CLCs were isolated from trifoliate orange. The six predicted proteins (PtrCLC1-6) had similar amino acids length, ranging from748to798, but they shared low sequence identity. Phylogenetic analysis revealed that the PtrCLCs were classified into two separate subgroup, PtrCLC4and PtrCLC6were more closely related to bacterial CLCs. In EcCLCA, the amino acids (S107, E148and E203) of the selective filter are involved in the interaction with Cl-. Comparison with EcCLCA revealed that the equivalent position of S107was P177in PtrCLCl, position of E148was A213in PtrCLC3, and position of E203was T260in PtrCLC4and S295in PtrCLC6, respectively. Real-time PCR analysis showed that PtrCLCs, particularly PtrCLC6, preferentially expressed in leaves of trifoliate orange. In addition, we analyzed the expression profiles of PtrCLCs in plants under nitrogen deficiency, NaCl and ABA treatments. Results showed that nitrogen deficiency inhibited expression of PtrCLCs, among which only the PtrCLCl reverted to the basal level when2mmol/L NO3-supplemented. In contrast, NaCl stress profoundly induced expression of the PtrCLCs particularly the PtrCLC2and PtrCLC4, both were also up-regulated by ABA treatment.4. A putative CCC encoding gene, PtrCCC, was isolated from trifoliate orange using homologous cloning. The PtrCCC was3,438bp long with an ORF of2,943bp, encoding a protein of980amino acids. The protein product was predicted to have11TMs. Phylogenetic tree revealed that PtrCCC was closely related to its counterparts in animal KCC transporters. Real-time PCR analysis revealed that PtrCCC preferentially expressed in tender tissues of trifoliate orange, such as leaf tip and root tip. In addition, its expression was up-regulated by KCl stress. PtrCCC was then over-expressed in wild-type tobacco to identify the gene function. Results showed that, under NaCl, KCl and NaCl+KCl stresses, the PtrCCC-overexpressing lines exhibited better growth phenotype than WT plants, which also had less reductions in root and shoot dry mass than that in WT under these salt stresses particularly under the KCl stress.更多还原