Transcriptional Analysis Of Young Cotton (Gossypium Hirsutum) Seedlings Under Salt Stress Via Solexa Sequencing

Salinity is one of the most important factors that affect producitivity of crops in the world. Improving the salt tolerance of plants is the main method to make the salinization land useful. Cotton is a model plant for studies of many biological processes. However, the growth and productivity of cotton are adversely affected by salt stress, especially at germination and young seedling stage. The salt tolerant mechanism of cotton has been investigated physiologically, including ion homeostasis,accumulation of osmolytes and antioxidants. However, molecular mechanism of cotton under salt stress is still unclear. Therefore, it is interesting to investigative the response of cotton to salt stress which will be helpful to improve the tolerance of the cotton to salt stress by molecular biology method.In the present study, an overall impression of gene profiles during the salt stress of cotton seedlings was acquired by deep sequencing. We found that many genes up- or down-regulated by salt stress from the sequencing result, which revealed the complexity changes of the transcriptome of cotton under salt stress. Then, we confirmed the sequencing result by realtime RT-PCR. We isolated the four LEA genes which is extremely up-regulated under salt stress, and comprehensively analyzed the expression levels of these genes in different tissues, and examined their responsiveness to salt stresses in yeast. The main results and conclusion were as follows:(1) Sequencing depths of 3 583 646 and 3 694 330 tags were achieved in the control and NaCl libraries, including 395 874 and 389 356 distinct tags, respectively. Then, tags recorded only once were wiped off, leaving 145 794 and 138 518 clean tags in each library. Overall, 75 500 (51.8%) and 72 077 (52.0%) tags in the solexa libraries treated without or with NaCl were matched to the reference genes respectively. We analyzed the most differentially regulated tags with a log2ratio >2 or <-2 using a greater statistically significant value (P<0.001) as well as false discovery rates (FDR<0.01), representing 223 up- and 317 down-regulated transcripts. The 45% of the transcripts is unknown. The genes participated in different pathway strongly suggested their regulating roles in the salinity adaptation in plants. (2) We confirmed the sequencing result by detected relative expression level of 22 genes by RT-PCR. Generally, the expression patterns of these genes showed agreement with the solexa data, which basically confirmed the reliability of our transcriptome analysis.(3) Transcript levels of 7 LEA genes were determined by qRT-PCR, the result suggested the 4 LEA genes we got from solexa data were most responsive to salt stress. Then, expression analysis of the 4 LEA genes was performed in leaves and roots. The result showed that the relative expression level of M19406 is much higher than TA24166₃635 and X15086, and the 3 LEA genes exhibited non-specific expression pattern. M19379 was likely root specific expression, the accumulation of its transcripts in leaves was barely detected even upon NaCl treatments.(4) Full-length of the 4 LEA genes were cloned by the means of RACE. We collected 4 LEAs from cotton, 18 LEAs from Arabidopsis and 14 LEAs from rice. Then, we performed a ClustalX alignment of these genes. The unrooted phylogeny tree result showed that the identified LEA groups are quite distinct from each other, and the 4 LEA genes in cotton were classified into different groups.(5) We expressed the 4 LEA genes in yeast, then analyzed the stress tolerance in solid and liquid YPG. The result suggested transgenic yeast with the 4 LEA genes have an improved tolerance to salt,osmotic and oxidative stress, which indicated the role of the 4 LEA genes in the adaptation of cotton to stress conditions. These results provide useful basic data for more in-depth analysis in Arabidopsis.