Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (14): 2651-2661.doi: 10.3864/j.issn.0578-1752.2016.14.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

The Function Expression of Salt-Tolerant Yeast Gene Halotolerance ( HAL1 ) in Cotton

MU Min, SHU Na, WANG Shuai, GUO Li-xue, FAN Wei-li, YIN Zu-jun, WANG Jun-juan, WANG De-long, YE Wu-wei   

  1. Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology/Key Laboratory for Cotton Genetic Improvement, Ministry of Agriculture, Anyang 455000, Henan
  • Received:2016-02-15 Online:2016-07-16 Published:2016-07-16

Abstract: 【Objective】 The objective of this study is to clone Saccharomyces cerevisiae halotolerance (ScHAL1) gene and transformed into cotton , explore the function of the gene in cotton, to further explore function of the salt-tolerant yeast genes in higher plants. 【Method】According to total length of mRNA sequence information of ScHAL1 in NCBI, the gene was cloned using RT-PCR technology from Saccharomyces cerevisiae As2.375, double enzyme digestion was made with XbaⅠand SmaⅠfor pBI121::GFP, and pBI121-ScHAL1::GFP fusion expression vector was constructed by In-Fusion technique. With weak auto-fluorescence upland cotton varieties, Y-2067, ZA-23 and GZ-2 pollen as materials, using the gene bombarding technique to study transient expression of cotton pollen. The expression vector pBI121-ScHAL1::GFP was transformed into cotton salt-sensitive material Zhong s9612 with gene gun in vivo conversion technology, T0 generation cotton genetically seeds were modified. Solution with 100 mmol·L-1 NaCl was used to test the salt resistance of seeds in the transgenic T0 generation seed germination experiment, molecular detection was carried out, and semal salt resistance of transgenic plants was analysis. 【Result】 ScHAL1 cloned from Saccharomyces cerevisiae As2.375 and ScHAL1 is 885 bp in length, which encoding 294 amino acids. After its sequence analysis, it was found that the largest proportion of the whole HAL1 protein is serine, and it is alkaline and positively charged and it is a hydrophilic protein. According to the results of protein secondary structure prediction, it is speculated that the structure of the protein function domain may mainly made up of random curl and beta sheet. Cotton pollen instantaneous expression results showed that after conversion of HAL1 gene, three land cotton powder green fluorescence were obviously enhanced, suggesting that the gene expressed in these three upland cotton pollen. With 100 mmol·L-1 NaCl, transgenic T0 generation seed germination ability obviously stronger than receptor s9612 selfing seed material, which preliminary showed that HAL1 could also improve the seed salt resistance. According to the gene nucleotide sequences, two pairs of primers were designed for molecular detection of T0 generation. Direct sequencing of PCR product was conducted and the sequencing results preliminarily evidence that the transgene is successful. With the semal salt resistance analysis, it was found that the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl and 400 mmol·L-1 NaCl were higher than the control, and the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl were higher than that in 400 mmol·L-1 NaCl. 【Conclusion】HAL1 gene was Successfully cloned from Saccharomyces cerevisiae, yeast HAL1 gene plays an important role in improving cotton salt resistance.

Key words: yeast, HAL1, upland cotton, pollen instantaneous expression, molecular detection

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