Study On The Functional Mechanisms Of HY2in Regulation Of Aluminum-mediated Root Inhibition In Arabidopsis

Soil acidification has been a major environmental factor limiting global foodproduction for a long time. It affects the production and the development of worldagriculture, and has been one of the most important factors that leads to massivedeforestation. Aluminum (Al) is the most abundant metal in earth’s crust. It occursprimarily as aluminosilicate minerals, most commonly as feldspars in metamorphicand igneous rocks, and as clay minerals in well-weathered soil, which shows notoxicity to plant. However, when the pH drop to5, aluminium ion will be released tothe soil. Its soluble ionic form (Al3+) shows phytotoxicity, which is characterized by arapid inhibition of root elongation. So aluminum toxicity and plant aluminumresistance research increasingly arouse people’s attention. When plants suffer fromaluminum stress, a large number of active aluminum stimulate plant signaling andproduce a series of signal molecules in the body. Then the related genes will beactivated and several self-defense mechanisms will be started. The research of genemicroarray and proteonomics show that Al stress can not only affect the transcriptionof the gene, but also affect the protein expression. Therefore, screening the Aluminumresistant genes is helpful to make further research on Al resistant mechanism.In this study, we used Colombia-0as the experimental material and cloned130genes which belong to various families such as MYB, NAC, C2H2and so on fromvarious tissues of arabidopsis. Through using In-Fusion clone technology, these clonedgenes were constructed into the expression vector pEGAD-3XHA-LUC respectivelyand formed35S::HA-LUC-CD fusion gene. Gene sequencing showed that thesequences were correct. By the method of Agrobacterium-Mediated GeneticTransformation, vectors were transferred into the wild arabidopsis Colombia-4. T1generations were screened by Basta and identified by the luminometer machine. Atleast2transgenic lines of every gene were got. Our team was obtained71differenttransgenic plants in the end.Through screening207transgenic plants received from our laboratory by theluminometer machine, i successfully got15genes whose protein expression level were induced by aluminum stress significantly. By reading various references andexploring experiment condition, i confirmed the concentration of buffer solution andAl3+were0.5μM and50μM respectively. Seven-day-old seedings were exposed to50μM Al3+stress solution (pH4.3). Then the35S::HA-LUC-CD fusion proteinexpression level was observed with the luminometer machine. The result showed thatfifteen genes whose protein expression level were induced by aluminum stresssignificantly. Among of them, nine genes such as AT3G09150(HY2) wereup-regulated and other six genes such as AT1G74870.1were down-regulated.To analyze the phenotype of these transgenic plants under aluminum stress, i foundthat the root of HY2transgenic arabidopsis was significantly longer than the wild type,which showed that the HY2transgenic arabidopsis was aluminum tolerant. When putthem under other stress, such as Cd, La and low pH, I found that there were nosignificant differences between them. Result showed that HY2transgenic arabidopsiswas induced by Al only. DNA detection and gene sequencing showed that HY2genehad been integrated into the genome of HY2transgenic arabidopsis and the sequencewere correct. Luminometer detection showed that35S::HA-LUC-CD fusion proteinwas expressed. The content of Al and callose in the root apice was significantly lowerthan the wild type.In conclusion, we proved that HY2gene was involved in regulating thephysiological process of root elongation which inhibited by Al stress. This resultwould be a new proof which is helpful to illuminate the Al resistant mechanism.