Study On The Relationship Between The Expression Of HrpZ_Psta Gene And Disease Resistance Of Transgenic Soybean

As an important oil, food and feed crops, soybean is known by people as a good diet in legumes substance with its rich nutritional value and health functions. But in recent years, the serious loss caused by the frequent outbreak of soybean disease and uncontrolled spread is worrying, devastating disease of phytophthora root rot of soybean, the general incidence could cut 30%-50%, high sensitive cultivar loss can reach 50%-70%, serious land will face loss.The loss that gray leaf spot disease brought to soybean production also let people can not bear to estimate, general susceptible variety reduce 10%-15%, serious when can reach 30%-50%. Although people have taken measures such as chemical control, but it did not play a significant role, and the pollution that chemical control bring to the environment and soil is not optimistic, so the cultivation of resistant varieties will become the most effective way of preferred choice.With the rapid development of transgenic technology, it provides a new way to cultivate resistant soybean varieties through genetic engineering. The hrpZPsta gene which isolated and obtained from pseudomonas syringae tobacco curing varieties were found to be an broad-spectrum disease resistance gene, hrpZPsta gene can be used as target gene transferred into plants, which is capable of encoding hrpin protein, inducing non-host plants allergic reaction, stimulating the plant to produce natural immune mechanism, and then make plants produce broad-spectrum disease resistance, also have no impact on the normal growth and development of plants.The hrpZPsta gene have been transformed into soybean receptor by the previous workers through Agrobacterium-mediated and pollen tube pathway method, and obtained the corresponding positive transgenic plants, then got T4 generation seeds after consecutive years of breeding and detection. When detected by molecular biology way, Real-Time Fluorescent Quantitative PCR(qRT-PCR) technique is used to determine target gene expression in the roots, stems, leaves and seeds of plants at the transcriptional level, which combined with the results of artificial inoculation identification test to observe its actual disease resistance performance, and analyze the relationship between the expression of exogenous gene hrpZpsta in soybean plant and plant disease resistance.The results obtained after research of three years are as follows:1. After the conventional PCR detection and Southern blot detection on T5, T6 transgenic lines JN29-705-21, JN29-705-22, JN29-705-15 and JL30-187, the results showed that, hrpZpsta gene has been successfully integrated into the soybean genome with a single copy integration, and it has stably inherited in the progeny of soybean transformation plants.2. QRT-PCR technique was used to determine the target gene expression in roots, stems, leaves, seeds of T5, T6 transgenic lines at the transcriptional level, results showed that, expression quantity of hrpZpsta gene were determined in different tissues, and high expression in leaves and roots, low expression in stems and seeds, which suggested that target gene hrpZpsta has expressed in transgenic soybean at transcription level.3. Hypocotyl infection method and spraying method were used to finish the artificial inoculation test on T5, T6 each transformed lines, the results of resistance identification showed, the resistant ability to phytophthora root rot and gray leaf spot disease improved more obviously compared with receptor plant. The resistant ability to phytophthora root rot and gray leaf spot disease of JN29 each strain improved from susceptible to the anti; and the resistant ability to phytophthora root rot of JL30-187 improved from anti to resistant, the resistant ability to gray leaf spot disease improved from mid- susceptible to mid-esistant.4. The correlation relationship between the expression which determined by qRT-PCR in different tissues of each strains and identification of resistance results was analyzed, the results further indicated that the expression of hrpZPsta gene in leaves was positively correlated to resistance ability of gray leaf spot,and reached the extremely significant negative correlation with the disease levels, it suggested the target gene expression in the leaf was higher, the lower the disease level, and its disease resistant ability was stronger, HrpZPsta gene which transformed into plants indeed effectively improved the resistant ability to gray leaf spot of plants.