Acquisition Of Transgenic ScALDH21 Cotton And Identification Of Transgenic Cotton On Salt Tolerance

Cotton is very important in China’s national economy.Molecular breeding technology can shorten the breeding cycle of cotton and improve cotton resistance with a single or multiple resistances.In this study,six cotton candidate varieties were selected as recipients in Xinjiang academy of agricultural and reclamation science,Manas Base of Early-maturing Cotton Research Group of Research Institute of Economic Crops,Xinjiang Academy of Agricultural Sciences and Korla Color Cotton Base of China Color Cotton Corporation.180097(from Agricultural Reclamation Academy of Sciences),Xinnongmian 1,109and ZY17-1(from Xinjiang Academy of Agricultural Sciences),Green Mian 2100 and Zongmian 2105(from China Color Cotton Corporation),respectively.ScALDH21 gene cloned from the extremely drought-tolerant Syntrichia caninervis moss was constructed into vector containing kanamycin and herbicide resistance marker,respectively.Color cotton and white cotton were transformed by agrobacterium-mediated spraying method.Combined with leaf selection mark,phenotype identification and PCR molecular identification,the transformed plants were selected out.The transform efficiency and the insertion position of target gene in cotton genome was analyzed.At last,the new transgenic ScALDH21cotton materials were obtained.At the same time,salt stress experiment was executed on T4 generation of ScALDH21 cotton(L92 and L96)in the seedling stage under the conditions of field and greenhouse conditions respectively.The chlorophyll content and survival rate of cotton with ScALDH21 gene and its receptor were measured under 0.4%,0.5%and 0.6%salinized soil,respectively,and the net photosynthetic rate,stomatal conductance,transpiration rate and instantaneous water use efficiency were measured.In the indoor pot experiment,MDA,POD,lignin,Na~+,K~+and Na~+/K~+values of different cotton lines under Na Cl(150 m M)treatment was calculated.The main results are as follows:(1)Two sets of vectors(p CAMBIA2300 and p CAMBIA3301)with kanamycin resistance or herbicide marker carrying exogenous ScALDH21 gene were constructed,and agrobacterium-mediated flower-spraying method was used to transform to the candidate main cultivars of Color cotton and white cotton in Xinjiang respectively.The statistics results indicated that the fruit setting rates of cotton after gene transformation were as follows:32.8%(180097),46.6%(Xinnongmian1),54.8%(109),29.7%(ZY17-1),18%(Green cotton 2100)and 10%(Palm cotton 2105).T0 generation seeds were propagated in Hainan.Cotton leaves were identified with 5000 ppm kanamycin or 2000 ppm glyphosate solution to screen resistant plants,and the detection rates of positive plants in the field were 8.3%(180097),6.9%(xinnongmian 1),0.6%(109)and 29.4%(ZY17-1).Color cotton was cultured in greenhouse and also was identified,and the final positive detection rate was 17.6%(green cotton 2100)and 19.3%(brown cotton2105),respectively.The positive transgenic plants were 180097(3 plants),Xinnongmian 1(4 plants),109(1 plant),ZY17-1(11 plants),green cotton 2100(1 plant)and brown cotton 2105(6 plants).The final transformation rates were 0.098%,0.170%,0.047%,0.288%,0.076%and 0.222%,respectively.(2)In this experiment,the T4 transgenic ScALDH21 cotton line of Xinnongmian-1 was used to evaluated salt resistance at seedling stage under field and greenhouse conditions.The survival rate and chlorophyll content of L92 were significantly higher than those of non-transgenic cotton under 0.6%salt stress.Under 0.6%salt stress,the net photosynthetic rate,stomatal conductance and transpiration rate of leaves of L92 line were significantly higher than those of its non-transgenic cotton.Under 0.5%salt stress,the instantaneous WUE of L92 and L96 was significantly higher than that of non-transgenic cotton.In greenhouse,MDA content of L92 was significantly lower than that of non-transgenic cotton under 150 m M salt stress.POD activity of L96 was significantly higher than that of non-transgenic cotton.Lignin content of L92 and L96 was significantly higher than that of non-transgenic cotton.The Na~+/K~+of L92 and L96was significantly lower than that of non-transgenic cotton.Based on the above analysis,this experiment verified that the salt tolerance of transgenic cotton with ScALDH21 gene was improved at seedling stage through the morphological and physiological indicators.(3)The insertion position of foreign gene in cotton chromosome of T4 transgenic cotton with ScALDH21 gene was determined preliminarily.The genome of cotton(Gossypium hirsutum)was downloaded from genome database Phytozome.Based on genome sequence files and annotation files,gene sequences were extracted using TBTools-GXF sequences Extract to obtain the local cotton gene sequence library.The 3’-end flanking sequence of ScALDH21 gene in cotton lines was used to search in the gene library of cotton.BLSATN comparison with the genome sequence of up land cotton showed that 607 bp was 99%similar to the A13 chromosome of up land cotton,and it is in the G089700 gene(dehydrogenase/reductase SDR family member 7 isoform X1),which was located at 4307 bp.