Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

The effects of ethylene on the HCl-extractability of trace elements during soybean seed germination

Background Ethylene is capable of promoting seed germination in some plant species. Mobilization of metals such as Fe, Cu, Mn, and Zn in mature seeds takes place when seeds are germinating. However, whether ethylene is involved in the regulation of soybean seed germination and metal element mobilization during early seed germination stage remains unknown. In the present study, seeds were treated with ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and double distilled H2O (ddH(2)0) treatment was used as control. Ethylene emission, ACC synthase (ACS) expression, ACS enzyme activity and Ca, Zn, Mn, Cu and Fe content in hypocotyls were qualified to analyze the relationship between ethylene and mobilization of these elements. Results The results showed that ACS expression, ACS enzyme activity and ethylene emission peaked at 1 and 7 d after sowing. AVG inhibited ethylene production, promoted the hypocotyls length, ACS expression and its activity, concentrations of total and HCl-extractable Zn, and HCl-extractable Fe in hypocotyls, while ACC caused opposite effects. AVG and ACC treatment had no significantly effects on total and HCl-extractable Ca, Cu and HCl-extractable Mn. Total Mn concentration was promoted by AVG at 1, 3, and 5 d significantly, while ACC treatment tended to have no significantly effects on Mn concentration. Conclusion These findings suggested that ethylene is at least partly involved in the regulation of soybean seed germination. Remobilization of Zn and Fe may be negatively regulated by ethylene.

Development of SNP Marker and CAPS Marker Linked to ACC Synthase Gene in Different Sexual Phenotypes of Cucumber / 生物化学与生物物理进展

Ethylene regulates sex expression in cucumber plants (Cucumis sativus L.). ACC synthase is a key factor during the ethylene biosynthesis. Pairs of PCR primers were synthesized corresponding to the conserved sequences of ACC synthase gene family. The 1 188 bp DNA fragment of ACC synthase gene (CS-ACS2) was amplified from genomic DNA of 8 different sexual phenotypes of cucumber respectively (GenBank accession number is DQ115884～DQ115886 and DQ115875～DQ115879). 8 SNPs have been identified by sequences analysis between 3 monoecious lines and 5 subgynoecious lines and gynoecious lines, which including 4 A←→G and 4 T←→C transition. Of these 8 SNPs, one locus is in intron and 7 loci in exons. Of the 7 SNPs located in exons, 3 SNPs are non-coding SNPs and 4 SNPs are coding SNPs (cSNPs) of which 3 induced changes of encoding amino acid of ACC synthase. The results of SNPs from subgynoecious lines and gynoecious lines suggest that single nucleotide mutation events of CS-ACS2 might be correlated with the development of subgynoecious lines and gynoecious lines in cucumber. Furthermore, CAPS marker C-MT705 was developed for identifying elite subgynoecious cultivar MT-705, which could be valuable in cucumber breeding. Besides, the SNPs and CAPS markers obtained in the study enriched molecular markers of cucumber.