Phloem Transport Properties Of YSF And Construction Of Amino Acid-guided Pesticide Carrier Screening System Based On OsAAPs And OsLHTs

Amino acid-pesticide is a compound that combines the natural amino acid with an insecticidal molecule,which can help the parent molecule to transport and accumulate in certain specific tissues in plants,where are suffering serious damage by pests.Previous studies have shown that the Glucose-fipronil(GlyF),a conjugate obtained by coupling the glycine and fipronil parent pesticide can be transported by the amino acid transporter RcANT15.However,it was found that its transport ability in the phloem was poor due to the presence of connecting arm between fipronil molecule and the glycine group.Simplified the structure of GlyF,we synthesized a new Acetic acid-fipronil compound named as YSF.The cotyledon absorption and competition inhibition test proved that YSF has the characteristics of amino acid transporter;Through the phloem conductance study,it was found that YSF has phloem conductance,and the YSF content detected in the phloem is about 30% of the culture solution.The results showed that RcANT15 also has functions in YSF uptake.The rhizomes of rice were sensitive to pests and diseases.Therefore,there is an urgent need for new types of transmissible pesticides to solve this problem.The 3 OsLHTs and 15 OsAAPs genes with high expression in rice rhizomes were selected to construct an amino acid-pesticide transporter screen system.Functional expression of OsAAPs or OsLHTs genes in Xenopus oocytes and patch clamp recordings were conducted to verify their affinity for a series of natural amino acids and targeted drugs.The results showed that the transporters of OsAAPs and OsLHTs have a substrate specificity that different transporters have different affinities for natural amino acids or amino acid-pesticides.In addition,in order to avoid the functional redundancy among the amino acid transport proteins,CRISPR/Cas9 single/multiple gene knockout techniques were used to produce 52 knockout mutants with different combination genes of OsAAPs or OsLHTs.50 Transgenic plants highly overexpression of OsAAPs or OsLHTs were also obtained by transgenic technology.Further breeding progeny plants with homozygote genotypes will be helpful for the identification of amino acid-guided pesticide carriers.