| Acidic soil (pH≤5.5) is one of the important limiting factors for agriculturalproduction, approximately30%of the global land area is acidic soil, and40%ofarable soil area is acidic. In acid soils, when the pH is reduced to5or less, Al whichwill be in the form of Al3+inhibits the growth and development ofplants (Kinraide et al.,1989). Therefore, Al toxicity is considered to be one of themain reasons for causing crop yields reduced and the decline in the quality in acidicsoils. Most plants have developed different aluminum tolerance mechanisms,the mostimportant mechanism is the secretion of organic acid from root chelated Altoxicity, much research on organic acid transporter family is mainly ALMT(malate transporter) family and MATE (citrate transporter) family. In this experiment,soybean Al-resistant varieties Jiyu70was selected as research materials,two MATE family genes GmAACT1and GmALF5were cloned by infusiontechnology, and Agrobacterium rhizogenes-mediated method was used to explore thefunction of two genes, in order to further clarify the two genes in the mechanismof aluminum tolerance in soybean. The main experimental results are as follows:1. RNA and DNA were extracted from Al resistant cultivar Jiyu70.GmALF5gene, GmAACT1gene and its promoter were cloned. Full length of GmALF5ORFwas1476bp,that of GmAACT1was1662bp. The two genes respectively encoded491and553amino acids and respectively contained with12and9transmembranedomains. The length of GmAACT1promoter sequence was1500bp,the promoter wasproved to have promoter activity by transient expression analysis in soybean hairyroots.2. The transcriptional expression level of GmAACT1and GmALF5genes wasstudied in Al-resistant varieties Jiyu70and Al-sensitive cultivar Jiyu62under Alstress (30μM Al3+) in the time course experiment (0h,2h,4h,6h,12h,24h) ofquantitative real time PCR. In Jiyu70,expression of two genes peaked at2h, andbegan to decrease in4h. The expression level of GmAACT1and GmALF5genes was3.3-fold and8-fold of those in Al-free treatment respectively in2h. In Jiyu62, theexpression of GmAACT1gene was0.4-fold of the Jiyu70in0h, although the expression reached the highest in4h, but compared with Jiyu70, the expressionof GmAACT1gene was lower in the whole process of Al treatment. However, theexpression of GmALF5gene in Jiyu62and Jiyu70had no significant difference in0h, but the expression of GmALF5gene handled rapidly rising in4hunder Al treatment, which was25-fold of Al-free treatment, the expression ofGmALF5gene was higher than Jiyu70in the whole process of Al treatment. Theseindicated that under Al stress, the expression of GmAACT1and GmALF5genes isdifferent in soybean Al-resistant varieties and Al-sensitive cultivar, but GmAACT1and GmALF5genes have a certain response on Al stress.3. GmAACT1and GmALF5genes were constructed into the green fluorescentprotein (GFP) by the method of Gateway, and transformed into protoplast extractedfrom Arabidopsis thaliana leaf, the results of subcellular localization showed thatGmAACT1and GmALF5proteins are located on the plasma membrane.4. Fusion expression vectors formed by GmAACT1and GmALF5genes andpCAMBIA3301expression vector were injected into soybean cotyledon node throughAgrobacterium rhizogenes transformation method, measuring citrate secretion ratesand staining hematoxylin. Overexpression of GmAACT1gene could enhance citratesecretion rate in soybean hairy root under Al treatment, the secretion rate was7.5times of wild type,3times of the vector, suggesting that the GmAACT1gene belongsto a member of the MATE citrate transporter family. Compared with wild type and thethe Vector, citrate secretion rate of overexpression of GmALF5gene in soybean hairyroot did not change obviously. Hematoxylin staining results showed thatoverexpression of GmAACT1and GmALF5genes can alleviate aluminum toxicity ofsoybean. These results indicated that these two genes possibly play an important rolein resisitant to Al in soybean, but regulatory mechanisms still need to be exploredfurther. |
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