The Thermotolerance Of Stamen Of Upland Cotton And Cloning And Fuction Analysis Of Genes Related To Heat Tolerance

High temperature stress is one major factor of limiting reproduction in upland cotton. The reproductive process of flowering in upland cotton is extremely sensitive to high temperature stress. The high temperature stress during developmental process of square often result in square growth delayed, structure of square deformed and pollen aborted. And finally, high temperture influences sexual reproduction and procedure of fertilization, resulting in reducting yield in upland cotton. A short high temperature occurs more frequently with the rising temperature in earth. Especially in Yangtze River valley, high temperature of above 35℃ occurs frequently in summer. At this stage, cotton is at flowering and boll’s peak stage. These high temperatures, occurred frequently, are extremely unfavorable for upland cotton (Gossypium hirsutum L.) upgrowth, resulting in square growth delayed, pollination and fertilization deformed, abortive boll increased and quality of fiber decreased. To screen and breed high temperature resistant upland cotton cultivars are very urgent for cotton production. Up to now, screening for high-temperature-resistant upland cotton at home and abroad mainly focus on the stability of cell membrane, osmoregulation, the metabolites of antioxidant system and photosynthetic system. Many parameters for screening high temperature resistant are not unify. These parameters reflect metabolite characteristics of cotton. The thermotolerance study on development of square and pollization and fertilization process remains unknown. Screening reproductive system resistant to high temperature in upland cotton is important for the production of cotton under high temperature condition.In this study, we select 9650Duanguozhi, Yankang1107, Sumian12 and Sumian16 as materials for experiment from 220 cotton cultivars depending on pollen grains germination results. These cultivars were tested to investigate the effect of high temperature and high culture temperature on pollen grains germination rate, the heterosis of pollen grains germination, the expression of heat shock protein gene in upland cotton stamens. We have established the parameters and culture temperature for screening stamen thermotolerace, and proved the basis theory for exploring the thermotolerance mechanism and screening resistant cultivars. The main results were as follows:1. The study on thermotolerance of pollen grains in upland cottonWe determined the pollen germination rate of 4 cultivars at 30℃、33℃、35℃、37℃ and 39℃ culture temperature. And then, we analyzed the relationship between pollen grains germination percentage and daily high temperature, culture temperature. We found that above 33℃ temperature influences the viability of pollen grains. The duration of daily high temperature was long, the influence of high temperature was serious. The difference among cultivars was obvious when the decline rates of pollen grains germination were the largest under the culture temperature 30-35℃. The culture temperature 35℃ is suitable temperature for screening thermotolerance cultivars. Less 41% decline rate of pollen grains germination percentage was a thermotolerance cultivar, more than 41% decline rate of pollen grains germination percentage was a thermosensitive cultivar.2. The study on pollen grains heterosis of thermotolerance in upland cottonThe pollen grains germination percentage of hybrid F1 was determined at three periods. The results showed that pollen germination percentage of hybrid F1 showed obvious mid-parent and transgressive heterosis after high temperature period. Before high temperature phase, hybrid F1 showed obvious advantage mid-parent and transgressive heterosis under 35℃ culture temperature. After high temperature phase, the retoration of pollen viability of hybrid F1 was stronger than parent at 30℃ and 35℃ culture temperature, and hybrid F1 exhibited obvious mid-parent and transgressive heterosis.3. Study on heat resistant mechanism of pollen grains in upland cottonWe observed the structure of anthers, pollen ultrastructure and pollen germination percentage before and after treated thermotolerance and thermosensitive upland cotton cultivars by high temperature. The results showed heat resistance of pollen related to the structure of anther during pollen formation process, organelles interior the pollen grains under high temperature stress. Under high temperature stress, the difference of pollen ultrastructure and anther structure is the main difference of pollen heat tolerance. The heat resistance of cotton pollen was regulated by the factors of stamen structure in development process, pollen ultrastructure.4. Cloning and expression analysis of two heat shock protein (HSP) gene in upland cottonTwo HSP genes in upland cotton, GhHSP2 (GenBank accessing number JQ756321) and GhHSP90 (GenBank accessing number JX843808), were cloned. The GhHSP2 was high homology with sHSP20 of other species, which belonged to the class of cytoplasmic Ⅱ HSP genes and related to heat resistance of cells. The GhHSP90 was high homology with HSP90 of other species, which associated with cell signal transduction, protein transport. The expression patterns of Gossypium hirsutum GhHSP2 and GhHSP90 were different in the leaves and stamens under high temperature stress. The expression of GhHSP2 and GhHSP90 in leaves displayed up, down and up patterns, relative expression of GhHSP2 reached the maximum at 1h. GhHSP2 preventing precipitation and aggregation of protein denaturation played an important role in to reduce injury by heat stress. The expression of GhHSP2 and GhHSP90 in stamens (anther) showed lifting pattern, relative expression reached the maximum at 12h, GhHSP2 was about 4-5 times compared with GhHSP90. GhHSP2 played an important role in stamen of upland cotton to reduce heat stress injury. GhHSP2, heat shock protein of cytoplasmic Ⅱ class, was an important molecule heat resistant protein in upland cotton stamen.From the study on thermotolerance of stamen of upland cotton and cloning and function analysis of genes related to thermotolerance, we found that the culture temperature of 35℃ could be used as screening heat tolerance for upland cotton cultivars, the 41% decrease in the rate of pollen germination could be used as reference index for screening heat tolerance cultivars in upland cotton, the mid-parent heterosis and transgressive heterosis were obvious above 30℃ culture temperature, the thermotolerance of pollen grains related to the structure of anther, ultrastructural pollen grains and the expression of HSP genes during pollen development, the GhHSP2 and GhHSP90 in upland cotton related to thermotolerance of upland cotton, overxpression of GhHSP2 and GhHSP90 could improve the heat resistance of the plant.