The Construction Of Inositol Sythesis Pathway In Vitro And Investigation Of Inositol Made The Effect On Wheat In Stress Tolerance

Inositol is a ring-shaped hexacarbon hexamelol.It is widely found in animals,plants,and microorganisms.The function of inositol involved in biofilm synthesis and signaling.Inositol also plays an important role in biological growth,development and response to environmental changes.Studies have shown that inositol can help fat and cholesterol degrade in animal body,and in plants inositol can regulate cell osmotic pressure to resist stress such as drought and high salt,help store mineral elements and participate in seed formation,which in turn has an important impact on crop growth and yield traits.Using synthetic biology to create a new way of inositol synthesis,exploring the role of inositol in promoting crop growth and development,resistance and increasing yield characteristics,has important theoretical significance and great application potential.The traditional production method of inositol is obtained mainly by plant extraction phytic acid hydrolysis,low content,high purification cost and expensive price,constructinol in vitro synthesis way,make use of a wider range of raw material sources,greatly reduce pollution and production costs,and develop the application of inositol in crop production,aquaculture,garden gardening and other fields which are only in the research and exploration stage.On the basis of the functional properties of 12 related enzymes,the in vitro high-efficiency synthesis system of xylose synthesis in vitro was optimized,and the effects of inositol on the growth and yield characteristics of wheat under the conditions of fertilizer reduction under field and indoor experimental conditions were studied,and the regulation of inositol on the salt resistance of wheat germination was detected.To lay an important foundation for further development of its application in wheat production.The main findings are as follows:1.Based on the analysis of bioinformatics and biochemical systems,it is constructed by xylose isomerase,xylulokinase,transketolase,ribulose 5-phosphate epimerase,ribose 5-phosphate isomerase,triose Phosphate isomerase,fructose-bisphosphatealdolase,transaldolase,fructose-1,6-bisphosphatase,phosphoglucose isomerase,D-myo-inositol 3-phosphate synthase,and inositol monophosphatase drove xylose transformed to inositol reaction pathway consists of 12 enzymes.2.The cloning,identification and screening of 12 enzyme genes have been completed.XK,TK,TIM,ALD,PGI,from Thermotoga maritima;XI from Thermus thermophilus;IPS from Archaeoglobusfulgidus.And these gene clones were recombined on p ET20 b,p ET28 a or p ET33 b,and successfully obtained p ET-20b-Ttc-XI,p ET-20b-Tm-XK,p ET20b-Ttc-TK,p ET20b-Tm-RPE,p ET20b-Tm-RPI,p ET20b-Tm-RPI,p ET33b-Ttc-TIM,p ET28a-Ttc-ALD,p ET28a-Tm-TAL,p ET20b-Tm-FBP,p ET20b-Ttc-PGI,p ET20b-AfIPS,p ET20b-Tm-IMP recombinant expression plasmids.3.12 of the above enzymes were Expressed in E.coli BL21(DE3),and purified by heat treated,and tested enzyme activity.The detection method of inositol was optimized,the sample was analyzed by using Bio-rad Aminex HPX-87 P analysis column to analyze the sample at 0.8 m L/min with water flow phase at 80 ?.4.An in vitro synthesis method of high efficiency from xylose to inositol was established.The reaction pathway was verified,the reaction temperature,buffer type and concentration,enzyme addition were optimized,and the conversion rate of xylose synthesized inositol was achieved by supplementation to reach 96.6%.In the debugging process of constructing the reaction path,the reaction of carbon rearrangement platform was applied above 70 ? for the first time,and Tris-HCl was found to have a better anti-Maillard reaction than Na2SO3.5.The regulation of salinity resistance and physiological mechanism analysis of inositol in wheat germination show that under the pressure of inositol,the content of propylene dialdehyde in wheat germination period has played a certain role in alleviating the damage of wheat under salt stress.6.Field tests show that inositol sprayed on wheat can increase the length and growth of wheat spike and increase the yield of wheat under fertilizer reduction conditions.In Henan Yuanyang test field using wheat varieties Zheng mai 7968,in that year's wheat encountered dry hot wind disaster,the non-fertilization area 1m M inositol leaf spray improvedwheat yield 17.04%,fertilization area 2m M inositol leaf spray improved wheat yield 32.25%.