Development Of Herbicide-resistant Transgenic Rice With Low Glyphosate Residues

Weeds are one of the main factors that affect the yield and quality of rice,and have threatened many agricultural systems.Glyphosate is a highly efficient,low-toxicity and broad-spectrum herbicide widely used all over the world.The combination of glyphosate-resistant transgenic crops and glyphosate is regarded as an important strategy of weed management in modern agriculture.At present,only EPSPS gene is used alone and can give transgenic rice sufficient and stable resistance to glyphosate.The disadvantage of this method is that glyphosate still accumulates and transports in rice plants.The gene application alone can not meet the needs of commercial application.There is an urgent need to dig out genes that can degrade glyphosate and cultivate transgenic rice with low glyphosate residue and high glyphosate resistance.There are two ways to obtain transgenic rice with low glyphosate residue and high glyphosate resistance:one is to screen genes that degrade glyphosate efficiently through mutation,and the other is to combine two genes with different mechanisms of glyphosate resistance.At present,there is a serious shortage of glyphosate-degrading genes with independent intellectual property rights and agricultural application value in China.Therefore,it is of great significance to explore glyphosate-degrading genes with independent intellectual property rights.In this study,three glyphosate oxidase genes BceGO-B3S1,BceGO-B4S7 and BceGO-B5R26,which were cloned by our research group and professor Liu ziduo’s research group,were studied based on the following two parts:first,the three glyphosate oxidase genes were independently transformed into geng rice zhonghua11 to explore the function and application potential of these genes in rice,and to cultivate transgenic rice with low glyphosate residues and high glyphosate resistance with agricultural production application potential.Secondly,BceGO-B3S1 and BceGO-B4S7 were respectively constructed on vector p TGH-3 containing glyphosate-resistant gene I.variabilis-EPSPS*,and transformed into geng rice variety Zhonghua 11 to cultivate transgenic rice with low glyphosate residues and high glyphosate resistance.Transformation of glyphosate oxidase gene alone into geng rice Zhonghua 11.The WBceGO-B3S1,WBceGO-B4S7,WBceGO-B5R26 and utr-ctp*were fused and constructed on p U130 vector,and geng rice Zhonghua 11 was transformed.The transgenic positive plants were sprayed with glyphosate at a dose of 3600 g/ha at seedling stage,and the transformed plants with WBceGO-B3S1 as resistance gene were blocked or died,while 50%and 80%of the transformed plants with WBceGO-B4S7 and WBceGO-B5R26 as resistance genes grew normally.The results indicated that transgenic lines with WBceGO-B3S1 gene were resistant to glyphosate to some extent,but not enough to tolerate 3600 g/ha glyphosate,transgenic lines with WBceGO-B4S7 and WBceGO-B5R26 genes had higher resistance to glyphosate,which is more suitable for cultivating transgenic rice with glyphosate resistance.From the transgenic lines with WBceGO-B4S7 and WBceGO-B5R26 genes,we selected two single-copy homozygous lines with stable expression of foreign genes and integration in intergenic regions,namely GOB4S7-14 and GOB4S7-93;GOB5R26-29and GOB5R26-36.The agronomic traits of T₄ GOB4S7-14,GOB4S7-93,GOB5R26-29 and GOB5R26-36 were investigated in the field without spraying glyphosate.GOB4S7-14 is closer to ZH11.The main agronomic characters of GOB5R26-29 are not significantly different from those of ZH11.At the same time,four homozygous lines and transgenic homozygous line ZY29 with single I.variabilis-EPSPS*gene were tested for glyphosate residues in aerial tissues after being treated with 3600 mg/L glyphosate for 1,5 and 10 days.Compared with ZY29,the glyphosate residues in GOB4S7-14 and GOB4S7-93 decreased significantly after being treated with glyphosate for 1 and 5 days,and the glyphosate residues in GOB4S7-14 were lower than those in GOB4S7-93.At the same time,compared with the control ZY29,the glyphosate residues in GOB5R26-29 and GOB5R26-36decreased significantly after 1 day of glyphosate treatment,and the glyphosate residues in GOB5R26-36 were lower.After 5 and 10 days,no glyphosate residues were detected in the two lines.According to the agronomic traits of transgenic lines without glyphosate spraying and the residual results of glyphosate,we initially selected GOB4S7-14 and GOB5R26-29 as candidate lines.Different concentrations of glyphosate were treated at seedling stage of T₄ GOB4S7-14 and GOB5R26-29.The results showed that the plant growth of GOB4S7-14 and GOB5R26-29 could not be obviously inhibited when treated with 3600 and 5400 g/ha glyphosate,respectively,which indicated that GOB5R26 had higher resistance to glyphosate than GOB4S7-14.The agronomic traits of T₄ GOB5R26-29 under glyphosate treatment were investigated.The results showed that compared with 0 g/ha glyphosate treatment,the agronomic traits of GOB5R26-29 did not change under 2700 g/ha.These results indicated that the transgenic line GOB5R26-29 may have higher application potential in agricultural production.Study on geng rice ZH11 with co-expressing of WBceGO-B3S1 and I.variabilis-EPSPS*.Codon-optimized WBceGO-B3S1 and I.variabilis-EPSPS*were transformed into a Oryza sativa subsp.geng rice variety Zhonghua11.Molecular detection and field agronomic trait analysis resulted in the selection of three homozygous lines with stable expression of single copy of the transferred genes integrated into the intergenic region.Under the treatment of glyphosate at the test amount in the field,T₄ and T₆ transgenic lines exhibited no differences in main agronomic traits from wild type ZH11.Under the treatment by 3600 g/ha glyphosate,the glyphosate residues in the aboveground tissues of the three candidate transgenic homozygous lines were significantly lower than those in the transgenic homozygous line with I.variabilis-EPSPS*alone at 1,5 and 10 days.The glyphosate residues in ZL147 was the lowest.Therefore,ZL147 is expected to be a variety resource for cultivating transgenic rice with low glyphosate residues and high glyphosate resistance.Study on geng rice ZH11 with co-expressing of WBceGO-B4S7 and I.variabilis-EPSPS*.The WBceGO-B4S7 and I.variabilis-EPSPS*were transferred into ZH11 by Agrobacterium-mediated genetic transformation,and the T₀transformed plants were treated with glyphosate of 3600 g/ha,thus obtaining high resistance positive plants.Through molecular detection and field agronomic traits analysis,we selected two single-copy homozygous lines ZX69 and ZX88 with stable expression of foreign genes and integration in intergenic regions.The agronomic characters of T₄ZX69 and ZX88 and ZH11 were investigated without spraying glyphosate,and ZX88was closer to ZH11.At the same time,glyphosate residues in aerial tissues of ZX69,ZX88 and ZY29 were detected after being treated with 3600 mg/L glyphosate for 1,5and 10 days.Compared with ZY29,the glyphosate residues in ZX69 and ZX88decreased significantly after 1 day of glyphosate treatment,and the glyphosate residues in ZX88 were lower than those in ZX69.After glyphosate treatment for 5 and10 days,no glyphosate residues was detected in ZX69 and ZX88.In greenhouse,the seedlings of T₄ ZX69 and ZX88 were treated with different concentrations of glyphosate.From the naked eye observation,ZX69 and ZX88 could tolerate 3600 and5400 mg/L glyphosate respectively,indicating that ZX88 is more resistant to glyphosate than ZX69.Therefore,ZX88 was selected as the target line.The agronomic characters of T₄ ZX88under different doses of glyphosate treatment were investigated.Compared with 0g/ha glyphosate treatment,there was no significant difference in agronomic characters when sprayed up to 2700 g/ha（3 fold recommended agricultural dose）.Transgenic strain ZX88 has a good application prospect in agricultural production.Both WBceGO-B4S7 and WBceGO-B5R26 can endow transgenic rice high resistance to glyphosate by degrading glyphosate,which have important application value in breeding high resistant transgenic rice with low glyphosate residues.GOB5R26-29 with single glyphosate oxidase gene,ZL147 with co-expression of I.variabilis-EPSPS*and WBceGO-B3S1,and rice ZX88 with co-expression of I.variabilis-EPSPS*and WBceGO-B4S7 have good agricultural application potential.