Saccharomyces cerevisiae As2.375 is purchased from Shanghai Industrial Institute of Microbiology. The pMD18-T Vector is purchased from TaKaRa. E. coli strain DH5α, expression vector pBI121:GFP are preserved by the research group. CCRI12, GK50, Y2067 and GZ-2 were conserved in the stress-resistance identification research group of Institute of Cotton Research of CAAS.

The RNA of Saccharomyces cerevisiae AS2.375 was extracted with the Ultra Clean Microbial RNA Isolation Kit (MO BIO) kit, whose concentration and purity was assessed by Nanodrop 2000 ultramicrospectrophotometer. The RNA was reversely transcribed into cDNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover kit. According to the obtained cDNA as the template, the full-length gene primer was designed using the Primer Premier 5 software, 5'-end: ScYCF1-F and 3'-end: ScYCF1-R (Table 1). The full-length sequence of mRNA was amplified using 2 × TransTaq-T PCR SuperMix. The amplification procedure was 94˚C 5 min, 94˚C 45 s, 55˚C 45 s, 72˚C 1 min, 40 cycles; 72˚C 10 min. The connected pMD-18T vector was transformed into competent Escherichia coli cells DH5α, and the positive clone was picked for sequencing verification. The physicochemical property of ScYCF1 protein was analyzed using the online-software ProtParam (http://web.expasy.org/protparam/), and the hydrophilic-hydrophobic property of protein was predicted by ProtScale

(http://web.expasy.org/protscale/). The secondary structure of protein was analyzed with SOPMA (http://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/MPSA/npsa_sopma.html).

The In-Fusion primer was designed on line (http://bioinfo.clontech.com/infusion) 5'-end InScYCF1-F and 3'-end InScYCF1-R (Table 1). The target sequence was obtained through amplification of the ScYCF1 plasmid as the template. The double cleavage sites Xba I and Sma I were chosen, and double enzyme digestion was performed on the expression vector pBI121:GFP. The fusion expression vector pBI121-ScYCF1:GFP was constructed with In-Fusion method, followed by recombinant clone verification and sequencing.

The pollen from upland cotton Y-2067, ZA-23 and GZ-3 were bombarded with portable particle gun GDS-80, and the gene expression was observed under laser scanning confocal microscope FV1000 (Olympus, Japan).

The powders and plasmids were treated as the way of Kong Jingjing [14] and the particle gun transformation in vivo was completed.

Four pairs of primers were designed for ScYCF1 gene sequence, 5'-end primers were ScYCF1-F1, ScYCF1-F2, ScYCF1-F3, ScYCF1-F4 and the 3'-end primers were ScYCF1-R1, ScYCF1-R2, ScYCF1-R3, ScYCF1-R4 (Table 1). DNA from transgenic plants was extracted with CTAB method for PCR amplification, and the program is 94˚C, 5 min; 94˚C 45 s, 58˚C 45 s, 72˚C 30 s, 40 cycle; 72˚C 10 min.

The germination experiment was performed on obtained T₀ seeds with the double walled filter paper method [14] , using 100 mM NaCl solution as treatment solution and water as control. Each group repeated for three times. The seeds were sent into the illumination incubator for incubation (condition of culture: 28˚C, illumination for 14 h; 25˚C, darkness for 10 h). The germination results were summarized after 7 d. Measured and compared the content of chlorophyll of the leaf discs, which obtained from the same position of 6 transgenic cotton and acceptor material CCRI12, were treated with 0, 400 and 600 mM NaCl and cultured at 28˚C under illumination conditions for 72 h to analyze salt tolerance of trans-ScYCF1 cotton.

The specific primer was designed according to the full-length sequence of Saccharomyces cerevisiae ScYCF1 gene retrieved from NCBI database (GenBank: NM_001180442.3). Gene amplification was performed using cDNA as template to obtain a nucleotide sequence with a length of 4584 bp (Figure 1), which was connected to vector for sequencing and turned out correct. ScYCF1 gene was inserted into a plant expression vector pBI121:GFP to construct green fluorescent fusion expression vector. Digestion by restriction endonuclease Bgl ІІ was verified to be properly inserted (Figure 2), indicating that the expression vector was successfully constructed and was named as pBI121-ScYCF1:GFP.

The results of bioinformatics analysis indicated that ScYCF1 gene encodes a

protein ScYCF1 with 1515 amino acid, the molecular formula of ScYCF1 protein was C7777H12253N2031O2199S54, with the molecular weight of 120.34 kDa and the isoelectronic point of 8.64, and located on the cell membrane. Amino acid composition showed that ScYCF1 protein leucine (Leu) takes up the largest proportion, 12%; which is followed by valine (Ile) and serine (Ser) taking up 8% each (Figure 3). With 156 positively charged alkaline amino acids (Arg + Lys) and 143 negatively charged acidic amino acid (Asp + Glu), the protein is alkaline and positively charged, the instability index is 36.15, with a half-life over 20 h, which belonged stable protein. The hydrophilicity and hydrophobicity analysis of ScYCF1 protein revealed that the quantity of hydrophobic amino acids in the protein much larger than that of hydrophilic amino acids (Figure 4), of which the GRAVY was 0.064 namely the whole protein was hydrophobic. The secondary structure prediction result (Figure 5) showed that in the protein there were 700 amino acids in α helical region, which constituted the main structure of the protein; there were 309 β-pleated sheets, accounting for 20.4%, 375 randomly coiled amino acids, accounting for 24.75%, and β-turn contains 131 amino acids, accounting for 8.65%. Thus, we could speculate that the structural and functional domain might be composed of β-pleated sheets and randomly coils.

Cotton pollen has auto-fluorescence phenomenon. Under excitation light at a wavelength of 488 nm, the auto-fluorescence of cotton pollen is green [14] . According to previous findings, three varieties Y2067, ZA-23 and GZ-2 with weak auto-fluorescence [14] were selected to collect pollen, and gene gun in vivo conversion technology was used to bombard the cotton pollen. The study found that

after the vector pBI121-ScYCF1:GFP was bombarded, the green fluorescence of pollens of three materials was significantly enhanced (Figure 6), indicating that ScYCF1 can be expressed in cotton pollen, which lay the foundation of using gene gun in vivo conversion technology to obtain transgenic salt-tolerant materials in the next step.

Choosing 20 grains of plump and uniform transgenic ScYCF1 seeds and CCRI12 inbreds was used for salt tolerance screening experiments. The seeds treated with 100 mM NaCl solution were taken as the experimental group, and those treated with water as the control group. The result (Figure 7) shown that under stress from 100 mM NaCl solution, the germination capacity of transgenic seeds was significantly higher than the control seeds, and the growth capacity of the root was significantly stronger than the control group. The root length of CCRI12 inbreds and transgenic seeds in water and under stress from 100 mM NaCl was measured respectively and it was found that under two kinds of treatment, the root length of transgenic seeds was longer than that of CCRI12 inbreds, indicating that the expression of salt tolerant gene ScYCF1 can not only enhance salt tolerance of cotton seeds, but also can enhance the growing ability of them.

Four pairs of primers were designed based on mRNA of ScYCF1 gene for

molecular detection of transgenic ScYCF1 plants. The length of amplified fragments of primers was 747 bp, 477 bp, 525 bp and 450 bp. The detection results were shown in Figure 8, expected band can be amplified from the transgenic plants and plasmid, while in the negative control CCRI12 inbreds there were no band amplified. Combining the sequencing results of PCR products (Figure 9), it was considered that the cotton was successfully transformed with ScYCF1 gene.

Salt tolerance experiments of leaf discs were conducted for six transgenic plants that were detected with correct molecular. After being treated with stress from

400 and 600 mM NaCl saline for 72 h, (Figure 10) the color of the cotton leaf discs gradually turned yellow, and the leaf margin turned white, while the leaf discs under 0 mM NaCl solution remained green; Under the same concentration of saline, the color and shape of leaf discs in ScYCF1 transgenic plants were more obvious than the control leaf discs. The chlorophyll content of the leaf discs processed was measured and results showed that under three kinds of treatment, the chlorophyll content of leaf discs in transgenic plants was significantly higher than that in CCRI12; but after treated with 400 mM NaCl, there wasn’t much difference in chlorophyll content between the two groups, so it is speculated that ScYCF1 gene of Saccharomyces cerevisiae can increase the chlorophyll content of cotton and plays a significant role in the tolerance of the cotton to salt stress.