| Wheat (Triticum aestivum L.) is an important food crop. It plays an important role for food security. Therefore, it has an important social and economic significance to ensure wheat production in high yield. High yield Wheat is determined by the following aspects:selection of high germination rate of seeds for sowing, planting techniques, field management techniques (such as anti premature aging, anti-lodging, etc.), control of pests and diseases (powdery mildew, stripe rust). How to guarantee high and stable yield of wheat research in China are focused on growing techniques, field management and pest control. But the study for the germination rate of wheat seed is relatively small. We always discover some varieties of low germination rate in the actual production of wheat. The risk of low germination rate of wheat is that not only farmers will increase the amount of seeding, but also seeds will vulnerable to be impacted from adverse conditions which can resulting in low wheat yields. To solve the problem of low seed germination of wheat, we must to find the reasons for low germination rate,such as biochemical, genetic and molecular aspects of cause.To better understand the mechanism of how wheat seed germination, we first use several wheat varieties as materials to determined the germination rate of all varieties. Then we measure several important physiological enzymes activity during the germination of wheat seeds, including CAT, POD, SOD and a-amylase. The results show that the germination rate of wheat was very significantly related to a-amylase activity; and related to the activity of POD, SOD and CAT is not very significantly. Then we have used isozymes electrophoresis to analysis a-amylase from different materials during different period in germination. The studies show that a-amylase patterns were different between species with high germination and low germination. At the molecular level, by extracting a-amylase gene mRNA from the seed endosperm during germination, and then by reverse transcription, PCR, cDNA cloning technology, we have analysis mRNA which have been extracted in the early period of germination of wheat seeds.Results are as follows:1.To solve the problem of low seed germination, we have to find out the reasons for the low seed germination. Through the germination test, we could screening of different varieties between high and low germination rate, then comparing their differences in physiological and biochemical. After doing that, we can analysis the causes of seed germination. Taking the conditions of our Lab's into account, we have do this germination test 7 days in the temperature of 20℃. We have discovered six varieties of wheat seed germination rate through the test. The germination rate of CN24 is the highest which correlation coefficient is 90.67%; MY11, CN17, CN19 which have higher germination, respectively, are 89.33%, 83.33%,86.00%; and CN27, CN23 which germination rate are lower, respectively, are 63.33% and 54.67%.2. On the germination of CN24/CN27 F2 population analysis found that the percentage of the number of material which have high and low germination rate is laws of Mendelian segregation of 1:3. And the difference is not significant. It shows that the germination rate in wheat may be caused by recessive genes. Then Wang Ping's study shows that the germination with the amylase activity by a-high, medium, low, to 1:2:1 for X2 tests. It is confirmed that high seed germination a-amylase activity was controled by a single recessive gene. we can initially speculated that the germination of wheat seed is effect by a-amylase activity. To confirm this, we need to do some further tests, such as the measuring of enzyme activity and molecular cloning, etc..The wheat seed germination is a very complex physiological process. During the process of expansion of seed in germination, the membrane system needs to repair and reconstruction, and the membrane repair is good or bad will directly affects seed vigor. And the seed germination contains the growth of embryo and the mobilization of storage product from endosperm and cotyledon. Seed storage material needs to be decomposition, then to be available in the embryo through the mobilizing to support the rapid growth of embryos. This process will require the rapid synthesis of a number of decomposition enzymes, such as CAT, POD, SOD and a-amylase. Through measuring the activity of CAT, POD, SOD and a-amylase during germination of different materials and analysis of the correlation, we could discover that:a-amylase activity and germination rate was very positive significantly correlated and the Correlation coefficient is 0.9743. And CAT, POD, SOD and the germination rate was positively significant and the correlation coefficients were 0.4588,0.4836,0.2362. So a-amylase is the most important pre-germination seed enzyme. This will consistent with the previous speculation.3.Through isozyme electrophoresis analysis a-amylase during the first three days of germination in all materials, we can knows which kind of isozyme is the most important enzyme in Seed Germination. Expressed during seed germination, we have found 5 kinds of a-amylase, respectively, A1, A2, A3, A4, A5. First day, A4 band have been co-expressed in the high germination rate materials, such as CN17, CN24, CN19, MY11; and CN23, CN27 common expression A5 band. The next day, CN17, CN24, CN19, MY11 increased A1, A3 two isozymes, which bands of which a high concentration of A1, and A3 with a low concentration of enzyme. CN23, CN27 have increased expression of the A1, A2, A3 three isozymes. We found that A3 is high concentrations while A1, A2 are density low. Because the germination with high rate didn't express A2, but A1 is high concentrations. Then the low germination rate species express Al with low concentrations and A3 with high concentrations. It shows that Al isozyme plays a major role during seed germination and A3 isozyme may plays a supporting role.4. We could analyze the a-amylase gene during seed germination at the transcriptional level, through cDNA clone of a-amylase gene from the endosperm of seed. Found that the a-amylase cDNA sequence of CN24 and CN27 have removed all of the introns and then connected all of the exon.There is no alternative splicing occurs. And we found that there are only a few bases mutant in the sequence between CN24 and CN27. But the mutation did not affect the important structures of a-amylase. So these mutation will not effect the germination. The difference of germination rate may be due to the different expression amounts. Non-coding region of amino acid mutation may cause the expression amounts was different. |
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