Studies On The Salt Tolerance Of Cotton Overexpressing An Apocynum Venetum DEAD-box Helicase Gene(AvDH1)

Cotton(Gossypium hirsutum L.) is considered to be the pioneer crop in the development of saline-alkali land. Although it is classified as a salt-tolerant crop, there are obviously variet al differences in response to soil salinity, and its growth and yield are severely affected in hig her salinity soil. Therefore, it is of agricultural importance to improve the salt tolerance of cott on. Helicases belong to a class of molecular motor proteins in yeast, animals, and plants. DN A helicases unfold duplex DNA and are involved in replication, repair, recombination and tra nscription, whereas RNA helicases catalyze the unwinding of the secondary structures in RN A and are involved in transcription, ribosome biogenesis and translation initiation. Recently,many helicase genes, especially DEAD-box genes, are reported to be key regulators of develo pmental processes and responses to diverse abiotic stresses, such as salt stress, oxygen levels,light, or temperature.We previously isolated a salt induced DEAD-box helicase gene, AvDH1(Accession num ber EU145588), from Apocynum venetum. The ligated construct(pBI121-AvDH1) was introd uced into cotton cultivar lu613 by pollen tube pathway-mediated transformation and A. tumefa ciens(strain LBA4404) mediated transformation and we obtained the salt-tolerant lines of cott on. In this study, we studied the salt tolerance of cotton overexpressing AvDH1 and salt tolera nce mechanism of transgenic cotton under salinity stress. The main results obtained experime ntally are as follows:1. PCR, Southern and Northern blotting analyses showed that the AvDH1 gene was integ rated into the cotton genome and expressed. Three transgenic lines 08-66, 08-87, and 08-26 w ere were used in further salt stress tolerance studies.2. In the growth chamber, no significant growth differences between non-transgenic and transgenic cotton expressing AvDH1 in normal conditions were seen. However, in the presenc e of NaCl, transgenic cotton had higher germination percentage and plant height than wild-typ e plant. Under salt stress, the AvDH1 transgenic lines had lesser ion leakage, lower level of M DA along with higher SOD activity, proline content and K+/Na+ ratio than WT plants. There were no significant differences among these three transgenic lines.3. Transgenic cotton lines expressing AvDH1 were field-tested under moderate(ECe 9.8dsm-1) and high soil salinity levels(ECe 15.6 dsm-1) at the Halophyte Garden Experimental Farm in Dongying, in 2013 and 2014. Averaged across 2 years, the transgenic lines 08-66, 08-87, and 08-26 produced 15.4, 17.9, and 15.9%, respectively, more seed cotton than WT in the moderate- salinity area and 23.9, 26.4, and 23.1%, respectively, in the high-salinity area. An i ncrease in boll numbers and boll weights contributed toward the increase in seed cotton yields of the transgenic AvDH1 cotton lines.4. To better understand the expression and functions of genes related to salt tolerance in t ransgenic cotton, we performed a digital gene expression(DGE) analysis between transgenic AvDH1 cotton and lu613. In nontransformed plants, 1359 DEGs were found following salt tre atment. In comparison, 1150 DEGs were found in transgenic plants after salt treatment, 751 g enes up-regulated in response to salt and 399 genes down-regulated. The results of qRT-PCR assays agreed with the DGE data, demonstrating the reliability of the transcriptome analysis.We found 35 transcription factors in the DEGs. Transcriptome profiling analysis showed that many key genes were involved in Phosphatidylinositol signaling system, peroxisome, ribosom e biogenesis in eukaryotes and RNA degradation,etc.5. We identified 161 putative RNA helicase genes in the genome of the diploid cotton sp ecies Gossypium raimondii. We classified these helicases into three subfamilies, which includ e the DEAD-box, DEAH-box, and DExD/H-box gene families. The AvDH1 protein shows a s equence identity of 74% to GrDExD/H4 and GrDExD/H30. Syntenic analysis revealed that G rDExD/H4 and GrDExD/H30 had syntenic relationship; Transcriptome sequencing data demo nstrated that GrDExD/H4 and GrDExD/H30 were expressed at high levels in all three samples and may play important roles in various processes of Gossypium raimondii.