Construction Of Mutant Library For TILLING Research And Screening Of Ae Gene In Zea Mays L.

Corn starch is a main starch source of human being. The starch is a major accumulated material of the corn seed, the maize starch is about 70% of the seed. The common corn starch is a mixture of amylose and amylopectin and the content is about 28% and 72%, respectively. Because of its special physical and chemical properties, amylose starch is a kind of high additional value starch and extensively applied in more than 30 fields as an important industrial material. However, the amylose content in common corn is generally between 22%-28% which limited its application in industry because of the expensive cost of extraction. The breakthrough of high amylose research is the discovery of the ae gene mutation body, the amylose content raised with ae gene. The effects of other modifying genes affect the starch content of endosperm. In order to improve screening efficiency and shorten breeding years, the research constructed TILLING Mutation Library by using EMS to induce maize pollen with high frequency single nucleotide polymorphisms. According to the sequence of ae gene we used PCR to amplify ae gene from mutant Mo 17. Through high-throughput screening with Lightscanner seven samples were acquired with SNP and the content of amylose is measured.The research induced 8902 and Mo 17 by using EMS pollination with the concentration of 0.0667% and the amount is 759 and 780, respectively. EMS had an impact on the growth of seed, thus the amount of seeds is 2960 and 3737, respectively. The leaf color and shape mutations were the main types in M1 seedling. Comparing to the non-mutation M1 seedling rate and adult rate were low, so chemical mutagen EMS greatly affected M1 seedling activity. The induction effects of various materials varied at the same EMS treatment concentration, thus it showed that various materials had different sensitivities to the using of EMS. The mutations of Mo17 were apparent to 8902. In M2 seedling, the leaf color mutation constituted the major part, including white seeding and yellow-green seeding. Because of the physiological damages of M1 kernel and plant by the EMS using, M2 adult mutations were very obvious. In M3 kernel, the ratio of mutation to contrast was near 3:1, and the germplasm character including white, yellow, sunken, scarcity was the main mutation type.Because phenotype was dominated by exons of gene and exons were more conservative to introns, 17 pairs of primers were designed according to the exons of ae gene. We used PCR to amplify ae gene from 512 Mo 17 mutants and the agarose electrophoresis showed that 17 pairs of primers were specialty. The PCR production of ae gene from 10ng/μl DNA by polling 8 DNA templates was screened by Lightscanner. According to the curve ofΔfluorescence to temperature, the different curve of mutation was found. Each sample of the curve contained should be amplified to ascertain the mutation sample. Finally we got 6 mutation samples from 4 curves of exons.The sequencing results showed that 5 mutations located at introns fields which had no effect on ae gene expression. In the exon 11 sequence of sample 92-5, the adenine was changed to thymine in the 8519 position, so the glutamic acid was changed to asparagines in the 358 position. The amylose content was 21.30% as 19.93% of contrast. The SNP of ae gene had minor effect on amylose content because of genetic background and gene interaction in the starch metabolism.Combining the chemical mutagen reducing high frequency mutations with high resolution melting curve screening by Lightscanner after PCR amplification, the research efficiently obtained SNP from mutation materials by EMS. It was a novel, high-throughput, low cost reverse-genetic method to help plant genome researches and improve maize inbred lines of high amylose.