Molecular Cloning And Expression Analysis Of Mulberry(M. Atropurpurea Roxb.) MaPGIP1 Gene

Mulberry tree is one of the perennial woody plants, belonging to Morus alba L. of Morus L.of Moraceae of Rosales. Mulberry leaves are the main source of food for the silkworm, and are important material base of’mulberry-silkworm-silk’ industry. In addition, Mulberry has a variety of development value, for example, it is an important ecological tree and also has important economic and medical uses. Recently more and more studies have been focused on comprehensive utilization of mulberry resources, and development of mulberry fruit industry has obvious advantages. Which is great significant to optimize the structure of mulberry industry and promote the sericulturist income. But mulberry fruit industry face a serious threat of sclerotium disease, Mulberry fruit sclerotiniosis is a kind of fungous disease and had happened in large areas of mulberry plantations for fruit use and caused heavy loss in mulberry fruit production.The cell wall is the first barrier that plant cells use to oppose the attack of pathogcns. Most phytopathogenic pathogens produce enzymes that are capable of degrading cell wall polymers, polygalacturonases (PGs) are the first enzymes to be secreted by pathogens when they encounter plant cell walls, PGs have been recognized as important virulence factors in pathogenic infection processes. Polygalacturonase-inhibiting proteins (PGIPs) that are located in the plant cell wall counteract the action of PGs, preventing cell wall degradation and therefore hampering the invasion process and the release of nutrients that is necessary for pathogen growth. The inhibiting activity of PGIPs directly reduces the aggressive potential of PGs. In addition, it causes PGs to form more long-chain oligogalacturonides that are able to induce defense responses, thereby indirectly contributing to the plant defense. PGIPs are important players in plant innate immunity and belong to the leucine-rich repeat (LRR) superfamily of proteins. In this paper, the study based on a genome-wide analysis of the morus genome database and used ’jialing40’(M. atropurpurea Roxb.) mulberry as the research object, the molecular characterization and functional analysis of PGIP from mulberry and transgenic MaPGIPl in tobacco will be research by using bioinformatics and molecular biology. The purpose is to lay the foundation and application for mulberry functional gene research and provide reference for breeding varieties resistant against pathogenic fungi through transgenic technology.Main research results are set out as below:1 Molecular cloning and bioinformatics analysis of MaPGIP1 gene from mulberryIn this test, a pair of specific primers was designed based on PGIP genes of mulberry (M. notabilis) in Morus Genome Database. The cDNA of ’Jialing 40’ PGIP gene was amplified from fruit by RT-PCR. The full-length cDNA of PGIP from ’Jialing 40’ fructus was obtained. Sequence analysis showed that the fragment contains an open reading frame of 1017 bp encoding 338 amino acid residues with a molecular mass of 37.9 kDa, named MaPGIPl. The gene sequence is submitted to NCBI for login, the accession number is:Gen Bank:KJ704112. This deduced protein has a pI of 6.65, Molecular formula of MaPGIP1 is C1717H2650N442O495S14, of which acidic amino acid (Asp+ Glu) accounted for 9.8%, basic amino acid (Lys and Arg) accounted for 9.5%, the highest percentage of leucine (13.3%). Unstable coefficient of the protein is 38.71, it shows that the protein is stabilized.a hydrophobic region of 26 amino acid residues in the N-terminal which was considered to be a signal peptide, and four potential N-glycosylation sites and contained 4 cysteine residues on N-terminal and C-temiani respectively, which involved in constituting the disulfide linkages. the center LRR structural domain is composed of nine tandem LRR motifs with the common sequence ’LxxLxLxxNxLS/TGxIPxxLxxL’ form the 76st amino acid, where L stands for a conserved Leu and x is any amino acid.Using Muscle software to conduct the multiple sequence alignment of MaPGIPl and other plant PGIP proteins, The results show that their homology is 50% to 65% comparing with Arabidopsis thaliana and Phaseolus vulgaris and 85% comparing with Morus notabili. Sequence differences mainly reflected on the non conservative sites.by constructing phylogenetic tree clustering analysis found that PGIP gene encoding amino acid sequences into two, respectively, monocotyledon and dicotyledon.The genetic relationship between MaPGIP1 and Prunus salicina and peach is very close.2 Expression analysis of MaPGIPl gene in various tissues and its responses to abiotic inductionThe transcript levels of MaPGIPl gene in different tissues (e.g. root, stem, bark, petiole, young leaf, old leaf and stipule) were detected and the expression in fruit with different development stages was analyzed by qRT-PCR. The results indicated that MaPGIPl gene had expression in various tissues, but expression levels had difference, the highest amount of gene expression in root, expressed in young leaves followed, the lowest amount of expression in the skin. In addition, MaPGIP1 gene showed down-regulated during the development of fruits, the expression levels of genes in front of the fruit development and mid significantly higher than that in the later. We also explore the MaPGIP1 gene expression level in mulberry leaves under salicylic acid (SA), abscisic acid (ABA), mechanical damage and low temperature(4℃) treatments, and the duration of the treatment was extended to 24 h. In the SA treatment, the expression level of MaPGIPl gene was up-regulated. The maximum relative expression values of MaPGIPl gene was shown at 3 h, which was about 25-fold up-regulated. Under ABA treatment, the expression of MaPGIPl gene was down-regulated. The MaPGIP1 gene expression level was reduced to one-fifth to one-sixth of the original. Under mechanical damage treatment, the maximum relative expression values of MaPGIPl gene was shown at 6 h, which was about 4.5-fold up-regulated. In the low temperature treat ment, the expression level of MaPGIPl gene was shown "down-up-down" trend, which was about 1.5-fold up-regulated at 6h and reduced to one-fifth of the original at 24h.3 Prokaryotic expression and Functional Analysis of MaPGIP1 GeneThe mature peptide of MaPGIP1 Gene was cloned into the prokaryotic expression vector pET28a(+), we construct recombinant expression plasmid pET28a-PGIP successfully.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).The recombinant protein pET28a-PGIP was expressed successfully under IPTG(final concentration 0.5mmol/L) at 28℃. It mainly appeared as inclusion bodies by SDS-PAGE analysis. Using for ultrasonic crushing methods to lysis the cell, then inclusion bodies was dissolved with 8mol/L Urea. Purifing expression protein with the Ni-NTA resin, the experimental results showed that high purity of the recombinant protein has been obtained. The purified protein was analysised by Western bolt. Finally, the MaPGIP1 protein with biological activity was obtained by sufficiently decrease the urea concentration in dialysate and by the method of dialysis refolding. The preliminary infection experiment of Sclerotinia mycelial inhibited to infect detached rape leaves by inoculating Ciboria shiraiana was tested. Results indicate that had centain inhibition effect of MaPGIP1 protein against infection of rapeseed leaves by Ciboria shiraiana at the early stage, and MaPGIP1 also relieved the disease symptoms caused by Sclerotinia mycelial. We determined the inhibition effects of MaPGIP1 on CsPG by DNS method. It turned out that the amount of D-galacturonic acid reduced gradually with the amount of MaPGIP1 protein increased, which CsPG degraded poly-galacturonic acid to generate D-galacturonic acid. It adequately show that MaPGIP1 protein can inhibit CsPG. MaPGIP1 partially inhibited CsPG with a pH optimum between 4.5 and 5.0, and a temperature optimum at 30℃, which can inhibit 45.5% of CsPG enzyme activity.