Cloning Of Several Important Maize Genes And Improving Amylose By Gene Transfer

Maize is an important food and feed crop in the world. We have made a great progress in maize traditional breeding. The advent of plant gene engineering and advanced molecular techniques for plant breeding provides powerful tools for genetically improving maize as to enhance its resistance to biotic stress and grain quality. The isolation and cloning of genes is the premise to plant gene engineering. We use the DD-PCR to isolate disease-resistance related genes from the maize inbred 48-2 which is resistant to Helminthosporium turcicum. Meanwhile, we use RT-PCR to clone the full-length cDNAs of maize genes sbe2b and sbel from maize endosperm total RNA with the primers designed according to the sequences of genes sbe2b and sbel in the GenBank. Two expression vectors were constructed using cDNA of sbe2b and sbel in antisense orientation, and transformation of maize was conducted. The main results are as following:The maize inbred 48-2 plants were inoculated by Helminthosporium turcicum when they grew to ten-leafed period. Total RNA was extracted from the leaves of inoculated plants and control plants 6 hours, 30hours and 54 hours postinoclution. 35 different cDNA fragments were produced by using reverse transcription-polymerase chain reaction (RT-PCR) fluorescent differential display. 13 of the 35 different cDNA fragments can be reproduced by PCR. 11 different cDNA fragments were identified by Reverse Northern blot hybridization and sequenced. The sequence of ddl(ZMCCDl) shares 80% similarity with that of CCD gene, the sequence of dd2(ZKIN) shares 85% similarity with that of SnRK gene. The sequences of ZMCCD1 (accession number:AY146585) and ZKIN (accession number:AY 146586) were submitted to GenBank.Total RNA was extracted from maize endosperm 15DAP(Days After Pollination) and cDNAs of sbe2b and sbel were obtained by RT-PCR. Endosperm-specificly-expressing vectors prEBE2 and prEBEl were constructed using cDNA of sbe2b and sbel in antisense oriental ion respectively.18 plates of immature embryos and 28 plates of embryogenic calli of maize inbreds501 and Xianzao17 were bombarded using mixed DNAs of expression vectors prEBE2, prEBEl and 35SIH3 by microprojectile (PDS-1000/He). After 3 times screening on medium with bialaphos in 2 months, 204 resistant calli were obtained from which 36 plantlets were regenerated and transplanted to flowerpots.By PCR, 8 of 10 plants can produce the same size 0.5kb specific DNA band as that produced from the vetor 35SIH3 by PCR; 5 plants have the same size 2.4kb specific DNA band as that produced from vector prEBE2 by PCR; 2 plants have the same size 2.5kb specific DNA bands as that from vector prEBEl by PCR. The sequences of the PCR bands were identified with that of genes Bar, sbe2b and sbel respectively. It indicated that genes Bar, sbe2b and sbel have been integrated into maize genome.We studied the effect of low temperature treatment on the efficiency of microprojectile (PDS-1000/He) transformation system using maize inbred 501 callus as receptor and Bar gene as selective gene. The result showed that it can enhance efficiency of the transformation if the callus was placed in 4"C for 10 hours and brought to 26'Cfor 4 hours before the callus was bombarded by microprojectile (PDS-1000/He)...