Differential Expression Analysis Of MRNA By CDNA-AFLP And Cloning Of Its Gene Fragments In Mulberry(Morus Alba L) Leaf Shape Mutant

The diversity of leaf morphological characteristics shows a wide adaptive feature in the environment. This distinct characteristic of leaves is not only important for plant production, but also aids the plants classification, such as that of in mulberry (Morus spp.). However, the expression and regulation of leaf morphological characteristics remains unclear. In the present study, the mulberry leaf shape mutant (Fengwei-Ichinose), and its wild type (Shin-Ichinose), were used to evaluate the regularity of leaf mutations and to identify the differentially expressed genes that respond for the changes of leaf shape. Fengwei-Ichinose was induced byγray of Co60 from'Shin-Ichinose' Histological observation was performed to analysize the leaf morphological characters, and the cDNA-AFLP was consducted to fish out the differentially expressed genes. The results are summarized as follows:1. The leaf index was distinctly different between the mutant and its wild type. That of Fengwei-Ichinose was 3.05, while that of the wild type "Shin-Ichinose" was 1.17, The leaves of Fengwei-Ichinose were much narrower and with transversal crimples, and thicker at leaf fully expanded stage when compared to those of the wild type. The constructure of leaf tissue, cell layers and epidermis, however, had no differences along the leaf length and leaf width direction between the mutant and its wild type.2. Total RNA was extracted from buds of mulberry using Trizol method with minor modifications. mRNA was isolated by PolyATtract(?) mRNA Isolation Systems (Promage). The cDNA was synthesized by SMART cDNA library construction kit (CLONTECH.). cDNA-AFLP was performed by polyacrylamide gel electrophoresis and silver staining. The results indicated that the quality of the RNA obtained by Trizol was very high. These set of techniques were applicable in mulberry.3. cDNA was digested by AseⅠand TaqⅠ, ligated by two annealed anchors, and then, amplified by primers complementary to the anchors with two selective bases at 3' end. The result of electrophoresis showed different primer combinations produced different banding pattern, while same banding patterns could be repeatedly obtained by a same primer combination Sequence of the specific between the mutant and its wild type showed the cloned fragments had the primer sequence and the two selective bases. These suggested cDNA-AFLP was of high reliability and repeatability.4. Totally 64 of combinations of eight AseⅠand eight TaqⅠprimers were used. More than 50 bands were detected from each primers combination, and approximately 3200 expression bands were obtained in sum. Of which,30 bands only appeared in the mutant or the wild type, were suggested may corresponsable to the leaf morphogenesis.5. Most of the bands between the mutant and its wild type were identicall, which demonstrated there was similarity between them. A few bands only appeared in one of the two materials, mutant or its wild type. This result suggested cDNA-AFLP is practiceal for gene different expression display.6. Fifteen of the different expressed gene fragments were cloned and sequenced. Two of them showed homological with known gene after BLAST on NCBI GeneBank. One fragment, obtained from Shin-Ichinose showed similarity to the UniGene of putative prolyl 4-hydroxylase, alpha subunit in Arabidopsis. Another fragment obtained from Fengwei-Ichinose showed homologous with hydroxymethyltransferase in Arabidopsis. The other 13 fragments did not show any homology with any known genes in GeneBank, suggesting they might be are new gene fragments responsible to mulberry leaf development, relating to the change of the leaf morphology in mulberry.7. It was the first time for cDNA-AFLP to be used to analyze mulberry. In the present study, the cDNA-AFLP technique system was established for screening the differentially expressional genes between mutants and their original type.Summarilly, From the above results we can conclud that the work of analyzing the mulberry mutants using cDNA-AFLP technique has developed a new strategy in the study of leaf development mechanism and cloning new genes in woody plant, which have been rarely reported up to now. The study established a solid basic for analysing molecular mechanism of leaf morphology development.