西藏青稞LEA3蛋白新抗旱基因的克隆与序列分析

Cloning and Sequence Analysis of Novel Drought-Tolerance Gene Coding LEA3 Protein in Tibetan Hulless Barley

以强抗旱青稞冬青8号和弱抗旱青稞品比14为材料,将幼苗经干旱诱导处理12 h提取总RNA,RT-PCR同源克隆到2个有差异的LEA3蛋白抗旱基因的全编码框序列。冬青8号、品比14的LEA3蛋白基因序列全长分别为661 bp和694 bp,其中,来源于品比14的LEA3蛋白基因编码框全长639 bp,编码含213个氨基酸残基的蛋白质,该多肽含有9个重复的、由11个氨基酸残基组成的保守基元序列。而来源冬青8号的LEA3蛋白基因与之相比较除缺少33 bp核苷酸外,另有6个碱基的差异,所编码的蛋白质也相应地缺少第4个保守基元序列,由8个重复的保守基元序列组成。二者在DNA与氨基酸序列上的同源性分别为94.38%和92.02%。结果证实抗旱性不同的青稞品种之间LEA3抗旱蛋白保守基元拷贝数有差异,推测抗旱蛋白结构的差异可能对植物的抗旱性有影响。

Late embryogenesis abundant （LEA） proteins were major group of proteins that were prominent in the stress response in various organisms including plants, algae, yeasts and bacteria. LEA3 protein was hydrophilic and accumulated in higher plants under conditions of extreme desiccation, during the last stage of seed formation, and during periods of water deficit in vegetative organs, in the present study, the most contrasting drought-tolerant genotypes were screened for comparing the differential sequences of gene coding LEA3 protein in Tibetan hulless barley, based on scoring of water loss rate （WLR）, maldondialdehyde and proline measured. And Dongqing 8 with the highest scores and Pinbi 14 with the lowest ones were selected for analyzing the differences between the genes coding LEA3 protein in Tibetan hulless barley. Total RNA of two selected barley cuhivars was extracted after treating with dehydration for 12 hours, and the first strand cDNA was synthesized by mRNA reverse transcription based on the primers designed according to the homologous sequence from Hordeum vulgate L. regiesterd in GenBank （No. X78205） . RT-PCR products of gene coding LEA3 protein were cloned to pMD18-T vector. The recombinant pMD18-T plasmids, inserted interesting gene, were identified by PCR and restriction digestion, respectively. Results of sequence analysis revealed that both genes coding LEA3 protein contained entire open reading frames. The ORF of gene coding LEA3 protein from Dongqing 8 was composed of 606 bp, contrasting with 639bp from Pinbi 14. Accordingly, the deduced LEA3 protein in drought-tolerant genotype, Dongqing 8, was composed of 202 amino acid residues, while that of 213 amino acid residues in drought-sensitive genotype, Pinbi 14. The deduced LEA3 protein in the most tolerant genotype contains 8 repeats of an 11-amino acid motif that form amphiphilic α- helical structure, accordingly it shares the same characteristics as the third group of LEA protein. Comparing with LEA3 protein of the most sensitive genotype, the deduced LEA3 protein in Dongqing 8 was absent of the fourth motif because of absence of 33 nucleotides. In addition, there was a mutation of 6 differential amino acid residues appeared between in Dongqing 8. The polarity of LEA3 protein in drought-tolerant genotype could be stronger than that in Pinbi 14, due to those 6 mutant amino acid residues. Respectively, both materials shared 94.38% and 92.02% similarity by DNA and amino acid sequence homological comparison. In the present study, differential number of repeated motifs was related to the most contrasting drought-resistant genotypes in Tibetan hulless barley. The differential hydmphilic capability of LEA3 protein could be related to differential number of repeated motifs, mostly to the polarity of mutant amino acid residues. Accordingly, it was suggested that differential configuration of drought-tolerant pmteins may be contributed to capability of dmught resistance in plants.