【作者】 李健；

【导师】 刘桂丰；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2010， 硕士

【摘要】 脂质转运蛋白是植物体内一种重要的防御蛋白,在抵抗和适应外界胁迫中有着重要的作用。本实验室已成功建立了在0.4 mol/L的NaCl溶液胁迫条件下刚毛柽柳根的cDNA文库,通过对柽柳cDNA文库克隆的随机测序,获得的ThLTP基因的cDNA序列,全长635 bp,其中5’非编码区129 bp,3’非编码区155 bp,开放阅读框(ORF)为351 bp,编码116个氨基酸。采用荧光定量PCR技术探讨了ThLTP在盐胁迫、冷胁迫、干旱胁迫、重金属胁迫以及ABA诱导条件下的表达模式,结果证明：ThLTP基因能够对NaCl、ABA, PEG和重金属隔(Cd)胁迫产生应答反应,显著呈上调表达,并且其在根中的表达量明显高于地上组织中的表达量；而冷胁迫则抑制了该基因在柽柳根中的表达。因而我们推测ThLTP是一个NaCl、PEG和重金属隔(Cd)逆境胁迫相关的基因,其表达受ABA诱导。将ThLTP构建原核表达载体,进行该基因的原核表达研究,建立了最佳原核表达体系,即30℃条件下0.2 mmol/L IPTG诱导5 h,重组菌株BL21 (pET32a-LTP)表达量达到最高。进而对重组菌株BL21 (pET32a-LTP)进行了逆境胁迫。结果表明,在NaCl和PEG胁迫条件下的生长曲线分析表明,重组菌株BL21 (pET32a-LTP)能显著提高大肠杆菌抵御非生物胁迫的能力,证实ThLTP是一个与抗旱耐盐相关的基因。为了进一步验证ThLTP在盐胁迫下的功能,将ThLTP基因用农杆菌介导法转化山新杨,共获得卡那霉素抗性株系20个,其中有10个株系经PCR验证为阳性。从10个株系中选择9个株系(S7、S9、S12、S13、S14、S15、S19、S30、S44)做进一步Northern检测,结果证明该基因已成功地整合到山新杨基因组中,并转录出相应的产物。将转基因山新杨和非转基因对照植株分别接种到含有0 mmol/L、50 mmol/L、100 mmol/L、150 mmol/L和200 mmol/L NaCl的培养基中培养20 d后,过量表达ThLTP的转基因山新杨植株生长状态好于对照植株。对200 mmol/L NaCl胁迫下叶片分化率、平均增重、生根率、盐害指数的综合分析得出,抗盐性从强倒弱排序：S14>S19>S9>S30>S12>S7>S44>CK。转基因植株叶片抗盐迫分析表明,0.8%NaCl胁迫3d、4d后各转基因株系叶绿素含量显著地高于非转基因对照植株叶绿素含量,其平均值高于对照的79.3%；各转基因株系相对电导率显著低于非转基因对照植株的电导率,其平均值低于对照的28.2%。由此可见,盐胁迫条件下,转基因山新杨受害程度明显小于对照,其中转基因株系S9、S14、S19较好,受到的伤害较小。转基因山新杨中过量表达ThLTP在逆境条件下对膜系统有保护作用,减轻质膜损伤,具有更强的抗盐胁迫能力。更多还原

【Abstract】 Lipid transfer protein is an important defense protein in plants, plays an important role in the resistance and adapt to stress. ThLTP from cDNA library was analyzed by bioinformatics, the gene has a total length of 635bp, the 5’non-coding region is 129 bp,3’non-coding region is 155 bp, the ORF (open reading frame) is 351 bp, encoding 116 amino acids. Its expected molecular weight is 22.76 kD, theoretical isoelectric point is 5.61.We studied the expression pattern by using real-time RT-PCR, and the results suggested that ThLTP was response to NaCl, ABA, PEG6000, and heavy metal septum (Cd) stress. it was up-regulated, and mRNA transcript level of this gene was higher in roots than leaves; while cold stress can suppressed the expression of ThLTP, the relative expression levels is lower than that of control, we speculated that ThLTP is a gene related to NaCl, PEG and CdCl2, induced by ABA.The ThLTP gene was inserted into the prokaryotic expressive vector pET32a and named BL21 (pET32a-LTP). And the highest expression system is 30℃,0.2 mmol/LIPTG induction 5 h. And then the recombinant BL21 (pET32a-LTP) were under stress. The results showed that NaCl and PEG stress on the growth curve analysis showed that the recombinant strain BL21 (pET32a-LTP) can significantly improve the Escherichia coli the ability to withstand abiotic stress, confirmed ThLTP was a drought and a salt tolerant genes.To further study the function of the ThLTP gene under salt stress, ThLTP gene were integrated into Populus davidiana Dode×P. bolleana Lauche via genome by Agrobacterium mediated method. It was totally obtained 20 independent lines with tolerance to kanamycin. Among the twenty, the expected 351 bp amplification product was detected in 10 lines. These nine transgenic lines (S7、S9、S12、S13、S14、S15、S19、S30、S44) were selected from the ten putative transformants for further analysis. The results of PCR and northern blot analysis indicated that ThLTP was expressed in the nine transgenic lines, and had integrated into the plant genome and could successfully transcript. The transgenic and thenon-transgenic plants were cultured on the medium with 0 mmol/L、50 mmol/L、100 mmol/L、150 mmol/L and 200 mmol/L NaCl for 20 days. Growth of transgenic plants was better than non-transgenic plants under Salt-stress conditions. The comprehensively analysis of leaf differentiation rate, average weight, rooting rate, salt injury index on the resistance to salinity indicated that the salinity resistance degree is S14> S19> S9> S30> S12> S7> S44> CK. Assay of salt stress of transgenic and non-transgenic plant showed that the chlorophyll contents of non-transgenic plants were significantly lower than those of transgenic plants under 0.8% NaCl after 3 d and 4 d, the average of chlorophyll contents was 79.3% higher than the control. The relative electrical conductivity of each transgenic line was significantly lower than that of non-transgenic plants, the average of chlorophyll contents was 28.2% lower than the non-transgenic plant. The experiments reflected that, under salinity stress, the transgenic plans suffered far less damage than control. Among them, S14 showed highest resistance and S9、S19 are inferior to it. The results also reflected that the over-expression of the gene in transgenic plants was beneficial for protecting membrane system.The expression of ThLTP gene strengthen the abilities of the salinity resistance.更多还原