Using Lxc As Expression Vector Containing Gna Gene For Inproving Insect-resistance Of Rice

Cyhodon dactylon L. (used name was Clavibacter xyli subsp, cynodontis) is an phloem vascular bundle-limited endophytic bacterium isolated from bermudagrass (Cyhodon dactylon L.) in the early 1980s, widely parasitize in Gramineae family such as rice, maize, sugarcane, oat and bermudagrass. And symbiosis can be proved by isolated itself from the plant tissues and organs, or amplifying its DNA with polymerase chain reaction. It is interesting that L. xyli subsp, cynodonti lives in vascular bundle of plant with high concentration and without obvious symptom. At present, transgenic technology is an effective biotechnology in plant breeding, which can lead foreign genes to express in plants, but it also has some problems including complex working and application security. Some researchers attempt to manipulate gene in endophytic bacterium, used as foreign genes expression vector. This method will not change genome of plant and be used in botanic transgene.Galanthus nivalis agglutinin(GNA) extracted from the bulb of snowdrop of Amaryllidaceae is acute toxic to sap-sucking homopteran insect pests including aphid, leafhopper, planthopper, and moderate toxic to chawing Lepidopterous insect pests.Our study focuses on inoculation method of Lxc in rice, characteristics of symbiosis of Lxc and rice, and effect on growth and development and yield of rice after inoculation. Based on these, pesticide endophytic bacteria Lxc/GNA12 was constructed for study on GNA expression in rice after inoculation, and obtained some beneficial results as follows:1. Lxc inoculated by leaf cutting at seedling stage can live in rice. Inoculation with Lxc did not effect growth and development and yield of rice, at the opposition, it could promote the heading and increase plant height of Mianhui725, and have yield increasing effect on Molixiangzhan USA.2. Immersing with Lxc taken as a method of inoculation was also feasible. Maturing seedling rate of mianhui725 was increased after immersing, however, effects on germination rate and maturing seedling rate of other varieties were unconspicuous. And 5.00D₆₅₀ is effective for immersing concentration of Lxc.3. Detecting of rice inoculated with Lxc by PCR technique, we could find Lxc live in stem and leaves of rice but not in mature panicles and grains. Fluorescence detection method could prove the result of PCR detection is right, at the same time, it proved foreign gene GUS can express in rice.4. Constructing vector pLGNA carrying GNA12 and transforming it to Lxc, we obtained pesticide endophytic bacteria Lxc/GNA12. GNA could detected in rice tissues when L. xyli subsp, cynodonti strain containing pLGNA12 was inoculated in rice.5. We inoculated Lxc/GNA in rice, and primarily identified resistance to brown planthopper and quantity of brown planthopper on rice. The results indicated that resistance to brown planthopper increased, quantity of brown planthopper on rice decreased.6. The results showed that the content of soluble sugar obviously increased in Lxc/GNA-inoculated rice, and, the content of soluble protein significantly decreased in Zaoxian D68 and of which the change was not significant in Molixiangzhan USA and Mianhui725. Amino acid contents(AAC) of inoculated-rice and non-inoculated-rice were determined by method of acid hydrolysis. The results showed that AAC of stems and leaves in Zaoxian D68 were both lower than controls with significant difference at 0.01 probability level, which in Mianhui 725 were both higher than controls with no significant difference level, and, which in Molixiangzhan USA were higher with no significant difference level and lower with significant difference at 0.01 probability level than controls, respectively.The results of investigation of three-year yield of rice proved that Lxc carrying foreign gene could improve some characteristics of rice. Identification of resistance to brown planthopper indicated that resistance to brown planthopper of inoculated-rice was higher than of non-inoculated-rice, but resistance identification should be further studied.Our study indicated that Lxc could be carrier of foreign genes expressing in plants, which will obtain target genes or correlative functions without change of genetic composing. So, this would be a new approach resolving security and technology of transgenic plants.