Rice Cryptchrome Gene RNAi Expression Vector Construction And Studies On The Transformation Of Rice

Using vectors pSPROK,pCAMBIA1300,pCAMBIA1301 and an intron from one of Arabidopsis thaliana genes as materials,an engineering vector named pSC1301-347 was constructed.Then,using pSC1301-347 as a tool vector,four RNAi and two antisense RNA expression vectors related to rice cryptochrome genes were constructed.They are pSC1301-347-Cryla,pSC1301-347-Cry2, pSC 1301-347-Crylab,pSC1301-347-Crylab2,pSC1301-347-antiCryl a and pSC1301-347-anticry2 respectively,of which the former four vectors are RNAi ones,and the last two vectors are antisense RNA ones.Through the method of Agrobacterium-mediated transformation,these expression vectors were used to transform japonica rice cultivar "Nipponbare",and finally more than 200 different colonial Gus positive plants were obtained.After sequencing analysis and the molecular assays of transformed plants,the inserted sequences of all the expression vectors were proved to be correct with the target gene fragments completely integrated into rice genome sequences.Based on the observation and analysis of T_o generation transformed plants,the preliminary conclusions were as follows:1)Cryptochrome genes had great effect on the seed-set percentage of ficeAccording to the performance of the four RNAi transformed plants which each had finished one life cirle,all the plants could normally headed and flowered,but basically bore no fruits.Transformed plants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9)did not get even one grain of seed,and only one or two plants from pSC1301-347-Cryla(V16)and pSC1301-347-Cry2(V19)bore a small amount of seeds while the seed-set percent for the control variety "Nipponbare" could reach as high as 20-30%.The reasons for the low seed-set percent were probably that besides the cold weather in winter and the abnormity in the growth of the transformed plants,because of the knockdown or restraint of the cryptochrome gene(s),there existed some metabolic disturbances during the rice young panicle differentiation,meiotic division or anther dehiscence and hence the anthers with normal vigor could not be produced finally. 2)Ovaries swelled,but did not filledAfter flowering,the rice spikelets were empty,or only the ovaries swelled with water-like substance in them and no accumulation of photosynthetic products. The transformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9) were especially like this.The reasons for this were probably that when two or more than two cryptochrome genes were restrained in expression,some obstacle(s) occurred during the biosynthesis or transportation for starch.3)Anthers were abnormalAnthers degraded,did not spread microspores with light color and a small number of microspores.The transformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9)were especially like this.The reasons for this were probably that cryptochrome genes were involved in some physiological or biochemical process of rice young panicle differentiation.4)The differences in plant morphologyUder the weak winter sun ligh of Fuzhou greenhouses,the transformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9)distinctly displayed a kind of morbidity.The plants were slightly taller than those from pSC1301-347-Cryla (V16)and pSC1301-347-Cry2(V19),with leaves seemingly longer and softer,and a little whiter and faded.5)The influence on floweringTransformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9) flowered later than those from pSC1301-347-Cryla and pSC1301-347-Cry2. Therefore,cryptochrome genes were related to rice flowering.It seemed that,the more the genes of cryptochrome were restrained,the greater the influence on flowering.Using rice mature and young seeds as material,the studies on rice embryogenic callus induction and the relevant genetic transformation were carried out.The results indicated that the callus induction rate for matue seeds,fresh mature seeds and young seeds was all＞81%,averaged 86.67%,with the induction rate for fresh mature seeds highest up to 96.87%.The resistance callus rate for mature embryo was 22.21%-40.71%,averaged 28.49%,for young embryo was 36.79%-43.21%,averaged 40%,with that for the young embryo higher than that for the mature embryo.The seedling differentiation emergence rate was 59.29%-80.43%with an average of 71.59%for the mature embryo,and 63.16%-72.73%with an average of 67.95%for the young embryo,showing no significant differences between the two kinds of embryos.The transformation rate(Gus positive rate)was 43.07%-81.08%with an average of 61.51%for the mature embryo,and 58.33%-76.67%with an average of 67.50%for the young embryo.The transformation rate for the young embryo was slightly higher than the mature embryo.The resistant callus rate,differentiation rate and transformation rate were different among different vectors to some extent, indicating that different inserted gene fragments had some effects on them.