Create Low-cadmium Waxy Breeding Materials By CRISPR/Cas9 System

Waxy rice is a natural viscous variety of rice.The appearance of the grain is milky and opaque.The starch contained in the endosperm is amylopectin with little or no amylose.Glutinous rice not only has rich edible value,but also plays an important role in the fields of food processing,medical treatment and industry.Cadmium is one of the inert heavy metals that is toxic to the human body.Cd in the soil is generally absorbed and transported by plants and eventually accumulated in the human kidney and liver organs through the food chain,threatening human health.At present,my country’s glutinous rice germplasm resources are scarce,and the varieties are relatively single,accompanied by the problem of cadmium pollution in rice.Therefore,it is necessary to create low-cadmium adsorption,green and safe glutinous rice germplasm resources.Previous studies have shown that the regulation of Wx expression can reduce the amylose content of rice,and the loss of OsNramp5 gene function can effectively reduce the adsorption of Cd by rice,which makes it possible to use molecular breeding methods to create low-cadmium rice breeding materials.This research mainly uses CRISPR/Cas9 technology to create new materials for low-cadmium adsorption glutinous rice breeding.The research content mainly includes the following two aspects.One is to knock out the OsNramp5 gene of the Gn to explore the absorption of Cd by the glutinous rice variety OsNramp5 mutants and the agronomic traits and quality characteristics of the plants.The second is to knock out the Wx and OsNramp5 genes of the indica backbone parents YX1 B and R498 materials to explore changes in mutant phenotypes and agronomic traits and quality.Through the above research,we hope to quickly obtain a new waxy material with low cadmium adsorption.The main findings are as follows:1.Research results of Gn OsNramp5 mutantOsNramp5 site-directed mutation was induced by Crispr/cas9 system,and 14 positive plants were obtained by genetic transformation.The results are as follows:(1)The Cd content in the seeds and leaves of OsNramp5 mutants is 0.082mg/kg and 0.089mg/kg,respectively,which is significantly lower than that of wild type,and both are lower than the safety standard of rice cadmium pollution in my country(0.2mg/kg).At the same time,the contents of Mn and Fe decreased significantly compared with the wild type,and the contents of Cu and Zn did not change significantly.(2)Mutant agronomic traits: plant height decreased,other traits such as flag leaf length,width,grain length,width,thousand-grain weight(average 20.708g),effective ear number,ear grain number,tiller number,seed setting rate(average 93.782%)There was no significant difference from wild-type Gn.(3)Quality identification: The mutant gel consistency,crude protein,amylose,amylopectin content,etc.did not change significantly,and the final viscosity decreased,indicating that the gene editing will not change the plant’s own quality and flavor characteristics,and may be chewed after cooking.Will be softer.2.Studies on the double gene mutants of YX1 B and R498Using the Crispr/cas9 system to induce Wx,OsNramp5 site-directed mutations,16 YX1 B positive strains and 21 R498 positive strains were obtained by genetic transformation.(1)Mutant molecular detection: Target sequencing analysis found that YX1 B and R498 mutations produced rich mutation methods,including insertions and deletions,and obtained double-target homozygous mutations in the T1 generation.(2)Phenotype identification: The endosperm of Yixiang 1B and R498 mutants is milky white and opaque waxy,with amylose content of less than 2%,which meets the standard of glutinous rice.The Cd content in grains and leaves dropped significantly below the cadmium pollution limit(0.2mg/kg).At the same time,the content of Mn and Fe decreased,and the content of Cu and Zn did not change significantly.There was no significant difference in chlorophyll content and wild type,and no typical symptoms of Mn and Fe deficiency.(3)Investigation of agronomic traits: The plant height and thousand-grain weight of the two mutants decreased significantly compared with the wild type.There is a slight fluctuation in a small range,but the difference is not significant.(4)Quality trait analysis: crude protein content,gelatinization temperature and amylopectin chain length distribution of mutants are similar to wild type.The amylose content was significantly reduced,the consistency of the gum increased to the standard of first-grade indica glutinous rice,and the viscosity of cold gum decreased,indicating that the mutant has excellent cooking and eating quality.In summary: In this study,one low-cadmium adsorbed waxy rice material and two new low-cadmium adsorbed waxy materials were obtained.Although it has a certain effect on plant height and 1000-grain weight,the plant grows normally and basically maintains the excellent characteristics of the material itself.Therefore,the methods and results of this study can provide a certain reference for the research on the cultivation of waxy materials with low cadmium adsorption.