Cloning And Expressing Regulation Of Expansin Gene From New Root Of Malus Hupehensis (Pamp) Rehd. And Primary Researching Of Its Physiological Function

Expansin can regulate the expansion of cell wall and control the development of the plant cell, then regulate the growth of roots. Malus hupehensis ( Pamp ) Rehd. var pinyiensis Jiang is one promising rootstock of apple trees and Ornamental Crabapples Cultivars in China. And that the roots are more important in the agricultural production. The formation and development of roots may be disclosed from moleculer biology, so we can regulate the growth of roots by the biologic technology. In this study, we isolated two full-length cDNA from new root of Malus hupehensis ( Pamp ) Rehd. and analyzed their bioinformatics. We researched the spatio-temporal expression of the two expansin genes, and the relationship their expression with endogenous hormones and ATPase. Furthermore we constructed the expression vector of pBI121-MhEXP2 and induced the expression proteins from E.coli loading MhEXP2 gene. The main results were as follows:1. Isolation of MhEXP geneBased on the sequences corresponding to conserved regions of expansin proteins from different plant species, two degenerate oligonucleotides were designed. With RT-PCR and RACE methods, two full length genes were cloned in root cDNA of Malus hupehensis ( Pamp ) Rehd. then named MhEXP1 and MhEXP2, with the accession numbers DQ538346 and DQ993160, respectively.2. Sequencing and structural analysis of MhEXP geneSequencing and structural analysis showed that the full-length of MhEXP1 consisted of 1111 bps with an open reading frame of 771bps that could code for a protein of 257 amino acids. The predicted MW and PI were 27.8kD and 8.9. And another full-length of MhEXP2 is consisted of 1320 with an open reading frame of 762 bps that could code for a protein of 254 amino acids.The predicted MW and PI were 26.8kD and 9.4. Eight cysteine residues and four tryptophan residues, which are supposed to be the characteristics of expansins, are conserved in both MhEXP1 and MhEXP2. The deduce amino acid sequences of the two genes have 56.98% identities. Phylogenetic analysis of other plant expansins indicated that MhEXP1 belongs to clade EXPA-V, while has more distant with GmaEXP1, OsEXP13, CsaECXP, MtrEXP, AtEXP11 and NtaEXP. MhEXP2 appears to belong to EXPA-I, which has more distant with PcEXP4, PcEXP5, PaEXP2 and MdEXP4. Thus, it is most likely that we have isolated the expansin gene of Malus hupehensis ( Pamp ) Rehd. By searching in PlantCare, both MhEXP1 and MhEXP2 have several cis-acting elements of auxin-responsive element. Furthermore MhEXP2 has a few special regulatory elements and motifs: MeJA-response motif ( TGACG mo tif ), low-temperature response element ( LTR ), heat stress response element ( HSE ). It implied that MhEXP2 may play a more role in the stress.3. Expression analysis of MhEXP geneNorthern blot showed that MhEXP1 is specially expressed in the root and that is regulated by IBA. MhEXP2 is expressed in the root and leaf, furthermore it is regulated by IBA too.4. The change of plasma membrane H+-ATPase and Ca2+-ATPase activities.Under treatment of IBA, PM Ca2+-ATPase activities were remarkably correlative with PM H+-ATPase activities that it's peak is earlier than PM H+-ATPase activities, so it indicated that Ca2+ as a second messenger may activate the PM H+-ATPase activities. The high PM H+-ATPase activities could decrease the pH of cell wall, so expansin should have a proper situation to play its function.5. The effect of endogenous hormones with IBA treatmentThrough analysis of endogenous hormones'content, it indicated that expression of expansins is not regulated by endogenous IAA, but remarkably correlative with the values of IAA/ABA.6. Construct the expression vectorIn order to determine the function of endogenous expansin MhEXP2 in growth and development of plants cell, a construct containing full-length cDNA of MhEXP2 gene in sense orientation driven by the constitutive cauliflower mosaic virus 35S promoter was assembled and introduced into tobacco plants. An expression vector carrying full-length cDNA of MhEXP2 gene in sense orientation with vector pET-30a(+) was constructed and introduced into E.coli., then induced expression proteins from E.coli loading MhEXP2 gene.