Dentification And Function Analysis Of Core Elememts In Promoters Of Abiotic-Stress Tolerance Genes Fras In Wheat

Abiotic stresses, such as drought, low temperature and salt, seriously affect wheat production. Cloning and functional studies of stress-related genes and their promoters not only lay the foundation for studying molecular mechanisms of stress tolerance in wheat, but also contribute for using molecular breeding method to improve wheat tolerance stress. Promoter servers as a switch in controlling gene expression, hence, to study promoter’s regulatory elements has an important significance on transgenic breeding.In our previous research, we have cloned TaFRAs (FRA:F-box Protein Related to Abiotic Stress) genes and their promoters and performed primary function verification. Based on these results,5’-deletion analysis of the three TaFRA genes’promoters (Pro_TaFRA1,Pro_TaFRA2,Pro_TaFRA3) was carried to discove core elements response to stress tolerance. According to distribution of the cis-elements predicted, different size fragments of each promoter have been amplified and fused to expression vector containing GUS. Through agrobacterium mediating infection of Arabidopsis thaliana, stable homozygote lines for each construct were successfully obtained. Finally, each homozygote line was stressed by ABA (100μM), drought (20% PEG-6000), salt (200mM NaCl) and low temperature (4℃) respectively. Through assaying GUS activity at different sampling times, induced atctivity differences of deletion fragments of three TaFRAs" promoters were systemly performed in this study. Major results are described as follows.1. According to results of promoters’ cis-elements predicited, the three promoters’ major difference in the kinds and the number of elements related to stress tolerance might lead to different induced activity in abiotic stresses. After stressed, all three promoters had induced activities with different induced intensity and speed. Under ABA stress, Pro_TaFRA1 reacted quickly, but Pro_TaFRA2 and Pro_TaFRA3 relative slowly; Pro_TaFRA3’s induced activity was the highest. Under drought stress, three promoters reacted slowly, and the induction activity of Pro_TaFRA1 was the strongest. Under salt stress,Pro_TaFRA1 and Pro_TaFRA2 reacted fastly,Pro_TaFRA3 relative slowly, and the induced activity of Pro_TaFRA1 was almost silimar to Pro_TaFRA3. Under low temperature stress, three promoters reacted slowly, and the induction activity of Pro_TaFRA3 was the strongest. In conclusion, Pro_TaFRA1 played a main role in dry stress, and Pro_TaFRA3 played a main role in both ABA and low temperature stresses, however, Pro_TaFRA1 and Pro_TaFRA3 worked together under salt stress. The induced activity of Pro_TaFRA2 was not significant comparing with others, and it only played a supplementary role after stressed. There existed greater differences among three promoters of TaFRAs under four abiotic stresses, which was consistent with those differences of cis-elements predicited in these promoters.2. In normal condition, GUS activity of Pro_TaFRA1-4 was the highest, followed by Pro_TaFRA1-1, and Pro_TaFRA1-3 was the lowest for TaFRAl promoter. These deletion fragments had induced activity under four abiotic stresses. Under ABA stress, the deletions driving GUS activity peaked at 0.5 hour with two times more than that in normal condition. Due to no differences of induced strength among the deletions, the cis-element response to ABA stress was not found. Under stresses of salt, drought and low temperature, all of these deletions performed activity, but Pro_TaFRA1-4 was significantly lower than the other constructs. Therefore, the cis-elements related to these three stresses existed between -830bp and-1393bp.3. Under normal condition, GUS activity of three 5’-deletion constructs of Pro_TaFRA2 declined along with the continuously trunction of the promoter from the 5’-side. Under four abiotic stresses, the induced activities of these deletions were below two times. After stressed by ABA, only Pro_TaFRA2-1 had strong induced activity, and the other two constructs’ activity was not obvious. Therefore, cis-elemen response to ABA may exist between-1793bp and -2639bp. After stressed by salt and drought, Pro_TaFRA2-1 and Pro_TaFRA2-2 had the same induced expression pattern, and the peak of GUS activity was also more about 1.5 times than that of no-stressed. However, Pro_TaFRA2-3’s expression pattern is different from the former two, and its induced activity is not obvious. Therefore, the main components in response to salt and drought were located between-799bp and -1793bp. In low temperature stress, three promoter constructs had the same degree of induced activity, and no significant differences of induced activity among the deletions were found. Therefore, the cis-element responsing to low temperature was not found.4. In normal condition, GUS activity of Pro_TaFRA3-2 was the highest and Pro_TaFRA3-4 was the lowest in the four delection constructs of Pvo_TaFRA3. Expression of Pro_TaFRA3-1 was similar to Pro_TaFRA3-3. These deletions had induced activity under four abiotic stresses. After ABA stressed, GUS activity in Pro_TaFRA3-4 had minor changes, but the others reached peaks at 24h, which was about three times of no stressed. The fomer three deletions’induced activities were significantly higher than Pro_TaFRA3-4. Therefore, the main cis-element responsing to ABA existed between -618bp and-1136bp. Furthermore, all the promoter constructs had induced activity to salt, drought and low temperature, and they had the same induced intensity and the same induced expression pattern. Those deletions had no reaction to salt, drought and low temperature, and the cis-elements responsing to these stresses were not found.