| Bamboo is widely distributed in the south of China.It is a kind of perennial Gramineae bamboo subfamily.As an important forestry resource in South China,bamboo plays a very important role in the national economy,China’s bamboo related industries have an economic value of about 200 billion yuan,which has an important contribution to the economic and social development.Temperature plays an important role in the geographical distribution,growth and development of bamboos.Bamboos are mainly distributed in the south of China where the annual average temperature is more than10 ℃,but it is difficult to grow in the north due to the limitation of temperature conditions.Temperature has become a key limiting factor in the project of South bamboo moving north,so it is of great significance to study the cold resistance of bamboo and expand its geographical distribution.The signaling pathway mediated by Inducer of CBF Expression 1(ICE1)is the most important low-temperature response signaling pathway in model plants.ICE1 as the core factor of this pathway increases the plant’s cold resistance by promoting the expression of downstream CBF genes at the transcription level.But in bamboo,cold stress-related pathways are still unclear,and no cold stress-related gene function has been verified in bamboo.Through transcriptome analysis,this study isolated the ICE1 gene induced by cold stress in bamboo,and then studied its function and obtained the following results:(1)A series of physiological and biochemical indexes of wild-type Moso bamboo under cold stress were analyzed.It is clear that ion leakage,malondialdehyde content and so on can be used as a marker to reflect the severity of cold stress in bamboo.(2)By analyzing the transcriptome data of wild-type moso bamboo before and after cold treatment,several genes involved in the response of bamboo to cold stress were screened,including ICE1,a key transcription factor in the cold resistant signaling pathway of model plants.(3)The Pe ICE1 gene was cloned and analyzed by bioinformatics.It was found that Pe ICE1 and ICE1 in barley,rice and other crops have high homology,both of them have the same conservative domain-HLH domain,suggesting that Pe ICE1 may have similar function with ICE1 in these crops.(4)Through the treatment of cold stress,ABA stress,drought stress and salt stress,the expression patterns of Pe ICE1 under various stress conditions were obtained by Quantitative Real-time PCR.The expression pattern of Pe ICE1 showed that Pe ICE1 could respond to cold stress,ABA stress and drought stress rapidly and positively,but Pe ICE1 could continue to respond to salt stress negatively.Compared with root and leaf,the expression of Pe ICE1 in stem was higher,which indicated that Pe ICE1 was mainly expressed in stem.(5)Using the protoplast of Moso bamboo as material,Pe ICE1 was transferred into the protoplast of Moso bamboo,and the subcellular location of Pe ICE1 was determined in the nucleus by microscopic observation.The activity of Pe ICE1 was confirmed to be suppressor by double luciferase reporter gene system.(6)Pe ICE1 was overexpressed in Arabidopsis by pollen tube transformation,and the transgenic Arabidopsis was identified.It was found that transgenic Arabidopsis had late flowering compared with wild type.It was found that overexpression of Pe ICE1 gene in Arabidopsis could improve its cold resistance.(7)Based on the regeneration system of Ma bamboo transformed by Agrobacterium,Pe ICE1 gene was overexpressed in Ma bamboo,and the transgenic bamboo was successfully obtained.(8)Compared with wild-type Ma bamboo,transgenic bamboo exhibited an increased cold resistant phenotype under cold stress.We measured the ion permeability,MDA content and chlorophyll content before and after cold treatment,and found that the physiological and biochemical indexes of transgenic bamboo were significantly better than that of wild-type Ma bamboo.We measured the expression of CBF3 gene in the downstream of ICE1 before and after cold treatment.We found that the expression of CBF3 gene in wild-type bamboo and transgenic bamboo increased after cold treatment,but the expression of CBF3 in transgenic bamboo was significantly higher than that in wild-type bamboo.It shows that overexpression of Pe ICE1 can improve the cold resistance of bamboo.In conclusion,this study isolated and identified the functions of the cold stress related gene ICE1 of Moso bamboo,provided the possibility of improving the cold resistance of bamboo through genetic engineering,and also provided the theoretical basis for bamboo breeding. |
