Studies On The Cloning And In Planta Transformation Of The Low-temperature-inducible Gene(Wap25) In Mulberry

Mulberry (Morus L.) trees are generally described as deciduous woody perennial species.Long-term natural selection and artificial selection produce plentiful accessions of mulberry resources. Some accessions show higher resistance to freezing, which can survive normally even at-30℃. They will serve as genetic resources for low-temperature breeding in mulberries. Presently, most researches about mulberry's frost resistance mainly focused on the measurement of some parameters for resistance, and have never been done in low temperature stress and/or low temperature response at the molecular level. Owing to the longer life cycle of woody perennial mulberries, less genetic population is available for scientific analysis. In this research, the responses of different mulberry varieties to low temperature stress are discussed, the genes induced by low-temperature were cloned, and the genetic improvement on the mulberries was performed at molecular level. This provides a scientific basis for further studies on resistance breeding of mulberry. The main research results are given below.1. Five mulberry species, Morus mongolica, with stronger cold resistance was screened out of thirty two mulberry races by acclimation at low temperature. Using its shoot as cutting for budding, the mulberry showed very strong cold-resistance after freezing acclimation at 3℃-0℃.2. Mongol mulberry was used as experimental material in this study. The stem cuttings at Magpie-mouth stage from this species were acclimated at 3℃for 48 h, from which the RNA was extracted and then reverse-transcribed into cDNA. The low-temperature-induced associated gene was cloned from Morus mongolica by using RT-PCR. The results indicated that the gene was 681 bp in length. Utilizing some softwares and databases for bioinformatics to analyze the cDNA sequence and its encoding product, it was proved that the sequence harbored whole ORF, encoding a putative product with 226 amino acids. The gene has been submitted to GenBank under the accession number DQ104333. The predicted values of pⅠand molecular weight for this product were 5.32,25.3 kDa, respectively. Thereafter, this protein was designated as WAP25. Among the 226 amino acids, hydrophobic ones hold a major portion, in which there are 35 alanine (15.5%),31 lysine (13.7%), and 29 glutamine (12.8%), respectively. These three kinds of amino acid residues account for 42% of total amino acids. It was found that a 11-mer amino acid motif (T**KAKEKA*D/E) was repeated for twelve times. Such repeat is just unique feature of group 3 late-embryogenesis-abundant proteins (LEA3), which was generally described as cold-induceible protein. In addition, this protein possessed typical structural features of signal peptide in the N-terminal region at position 1-33 amino acids, which implied that Wap25 was a secreted protein.3. The DNA fragment of Wap25 was cloned into a prokaryotic expression vector, pET-28a, generating recombinant plasmid, pET28/Wap25, which was then transformed into E. coli BL21. SDS-PAGE electrophoresis assay confirmed its effective expression as a specific band of-28 kDa in size, indicating that the size of the expression product was in consistent with the anticipation. And western blotting assay also showed the same result.4. The DNA fragment of Wap25 was cloned into a binary expression vector, pIG121-Hm, generating recombinant plasmid, pIG121/Wap25, which was then transformed into Agrobacterium tumefacie LBA4404, obtaining recombinant Agrobacterium tumefaciens strain LBA4404/pIG121/Wap25, which was resistant to four kinds of antibiotic (Kan, Hm, Str, and Rif). This recombinant Agrobacterium was used to infect leaf disks of Petunia. In MS medium containing Kan and Hm, green sprout appeared in parts on the leaf disks, and then adventitious bud was developed. 60 days later,148 transformants resistant to both Kan and Hm were obtained. The genomic DNA was extracted from transformants as template in a PCR assay. The results showed that among the 8 transformants,4 transformants were confirmed to be positive, suggesting Wap25 gene has been integrated into the genome of Petunia. The transformants efficiency is 11.48%.5. The mulberry seedlings with forming main leaves were infected with recombinant strain A-110 (LBA4404/pIG121-Hm) by in planta transformation. In the experiment for detecting remained Agrobacterium tumefaciens, it was observed that, there was a certain amount of Agrobacterium cells on the infected leaves. No VirA gene was detected in the remained cells by PCR amplification, which indicated that there was no Agrobacterium tumefaciens residue in the infected seedlings. In a further study, it was shown that many deformed leaves on the infected seedlings. Positive signals in deformed plantlets were found by PCR-Southern amplification, Southern blotting and GUS satining, which suggested that foreign gene,β-glucuronidase(GUS), can also be transferred into mulberry seedlings by in planta transformation.