| The RWP-RK gene family is a plant-specific family of transcription factors.They are not only key regulators of plant nitrogen response and gametophyte development,but also their high expression can regulate a plant’s special reproductive mode-apomixis.However,the studies on RWP-RK family genes are mainly focused on non-food crops,and less on some important food crops,such as rice.In this study,based on transgenic means and genome editing techniques,the overexpressed transgenic lines and knockout mutants of RWP-RK family genes were obtained.By analyzing the overexpressed transgenic rice,we understood its influence on the main agronomic traits of rice,laying the foundation for further explanation of the function and genetic improvement of the RWP-RK genes in rice.The main results were as follows:1、Construction and transformation of overexpression vectors.Through rice bioinformatics related websites,12 rice RWP-RK family genes（named OsRWP1-OsRWP12）were found.In this study,the CDS sequences of 6 genes were selected for artificial synthesis,and the corresponding overexpression vectors driven by the ubiquitin promoter were constructed,named OR1,OR2,OR5,OR6,OR9,OR11-1 and OR11-2.Agrobacterium-mediated genetic transformation on the materials of indica rice Minghui 86 and japonica rice Xiushui 134,among which OsRWP1（OR1）,OsRWP2（OR2）,OsRWP6（OR6）,and OsRWP11（OR11）all obtained positive transgenic plants,However,OsRWP5（OR5）and OsRWP9（OR9）did not detect the transgenic events.q RT-PCR analysis found that the corresponding target genes in the transgenic positive lines were significantly expressed.2、Investigation of agronomic traits.Genetic analysis of T1generation transgenic plants found that the overexpression lines of different genes had different inheritance laws.The separation of transgenic components in the transgenic offsprings of OR1,OR2,and OR6 completely conformed to the Mendelian law of inheritance,indicating that high expression of OsRWP1,OsRWP2,and OsRWP6 genes had no significant effects reproductive development in rice.The OR11 transgenic offspring had a low separation ratio of transgenic components,which did not conform to the Mendelian inheritance law.The Kas square analysis found that it conformed to the2:1 separation rule.Further phenotypic analysis of the main agronomic traits found that,compared with the wild-type control,there was no significant difference in the heading stage and development of floral organs（inner and outer lemma）in the overexpression lines of 4 genes.However,the pollen fertility and seed setting rate of OR11 transgenic positive plants changed significantly.The pollen black dyeing rate was46.0%-48.5%and the seed setting rate was reduced by 25%-49%.3、Construction of CRISPR/Cas9 knockout vectors.Each two target sites were selected from 7 RWP-RK family genes to construct knockout vectors using the CRISPR/Cas9 genome editing technology,then transformed into the rice material.At present,all the 7gene knockout vectors have obtained positive transgenic plants.Sequence analysis of the target sites revealed that the target sites of genes OsRWP1and OsRWP5 were mutated.There were 5 mutation types in OsRWP1 and OsRWP5. |
