Isolation Of Genes Involved In Quality And Genetic Improvement Of Starch Quality Of Seeds In Wheat

In this study, a cDNA library was constructed using wheat seed tissues at 12 d after pollination. Plasmid DNAs of 10,000 clones randomly picked out from the library were prepared. The preparation of high density filters were made with the Biomek 2000 HDRT system, and then hybridized separately with three probes prepared by reverse transcription of RNA of unpollinated ovary, embryo and endosperm. Based on the hybridization results, 800 clones expressed in embryo and/or endosperm were chosen for further analysis of expressed sequence tags (EST). Finally, 216 different genes were identified preliminarily. Of them, 24 (11.5%) were considered identical to known wheat genes, 122 (56%) were identified as putative new plant genes which may be involved in seed storage proteins, biochemical metabolisms, development, and other biological processes of seeds, while 70 (32.5%) sequence identities could not be determined. Furthermore, to obtain transgenic waxy wheat lines, the GBSSI gene of wheat was screened out from the cDNA library of seeds by cDNA microarray. DNA blot analysis indicated that there are three GBSSI genes in wheat genomes, and RNA blot hybridization showed that GBSSI mRNA was accumulated in seeds after pollination. By RNA silencing strategy, 683-bp sense and antisense fragments in reverse orientation separated by an 150-bp intron driven by maize ubiquitin1 promoter were cloned into pCAMBIA 3300. By Agrobacterium-mediated wheat transformation (Cheng et al, 1997), four transgenic plants (cultivar Yangmai 10) were identified by PCR and leaf painting assay analysis. The four lines showed different levels of amylose in endosperm by iodine staining. The main results are as follows: 1,Construction of wheat cDNA library cDNA library was constructed using the seeds of 12 d after pollination of PH82-2-2. Total RNA of wheat seeds at 12 d after pollination was extracted from 5 g seed tissues by using the total RNA isolation system (GIBCO, BRL), and the process of cDNA library construction was referred to the manufacturer's protocols (TaKaRa, Dalian, China). The reaction product was fractionated by Sizesep 400 Spun Column, and the fractions containing cDNA larger than 500 bp were collected and ligated into pBluescript ⅡSK(+) at the NotI and EcoRI sites (Stratagene). Then, the ligation products were transformed into E.coli JM109 (TaKaRa, Dalian, China). 2,cDNA microarray analysis For plasmid DNA preparation, ten thousand clones were picked out randomly from the cDNA library. After that, the denatured DNA were spotted onto nylon membranes using the Biomek 2000 HDRT system. Three probes were prepared by reverse transcription of 10 μg total RNA of ovaries at the anthesis, embryos and endosperms at 12 d after pollination with RAV-2 reverse transcriptase. Overnight hybridization was made with three different probes, respectively. 3,Analysis of differential hybridization Hybridization signals were read manually and classified into four kinds: no, weak, moderate and strong signals represented by 0, 1, 2, 3, respectively. Using basic programme, 8 different results were produced, namely clones expressed in only one tissue, in only two tissues, in all three tissues and undetectable in all kinds of tissues. 4,Sequencing analysis and isolation of genes involved in wheat quality Based on hybridization results, eight hundred clones were chosen and sequenced with ABI 377 DNA sequencer (Perkin Elmer, USA). Analysis of DNA or protein homology in GenBank was performed using the program Blast. The results indicated that 216 different genes were identified preliminarily. Of them, 24 (11.5%) were considered identical to known wheat genes, 122 (56%) were identified as putative new plant genes which may be involved in seed storage proteins, biochemical metabolisms, development, and other biological processes of seeds, while 70 (32.5%) sequence identities could not be determined.