Studies On The Heat Tolerance And Gene Of Heat Stress Response Related In Cucumber

As one of the environment stress factors, high temperature is often suffered in the protected field at summer, and the heat injure of cucumber, a major vegetable cultivated in protected condition, has become one of the main factors disturbing its normal growth, so how to improve the heat tolerance of cucumber has been one of the important research target in cucumber breeding. In this dissertation, the physiological mechanisms and molecular biology of heat tolerance were studied in cucumber, in order to deeply understand the mechanism of heat tolerance. The follows were researched:1 Studies on the heat injury symptom and selection of heat tolerance germplasmThe heat tolerant cultivar Poinsett 97 and the heat sensitive cultivar Boothbyls Blonde were used the materials of examination. The seedlings were treated with 42℃for 24h. The heat injury symptom of the cotyledon, leaf, hypocotyl, young leaf and seedlings were observed. The classification clear criterion of injure symptom were put forward in the cucumber seedling. The results are important for detecting heat injury and identifying heat tolerance.In order to screen out the heat tolerance material of the cucumber during the seed germination, we had compared that the germination ability under 28℃, 38℃and 42℃at different cucumber cultivars. The results shown: the heat tolerance of Xishuangbanna cucumber, Poinsett 97 and Beijinjietou was better than HH1- 8-57 and Erzaozi.In order to select out the heat tolerance material of the cucumber, we had compared that the heat tolerance in the field and the heat tolerance of the cucumber seedling under 42℃for 24h in different ecotype cucumber cultivars. The results shown: it was identified that five cultivars were heat tolerance and two cultivars were heat sensitive. The results was enrich for the material in the study of heat tolerance and heat injury in the cucumber. It is play a role in solve the problem of heat injury in the cucumber production.2 Study on the physiological parameters of the heat tolerance in cucumberThere cucumber varieties (Poinsett 97, heat tolerance variety; Boothbyls Blonde, heat susceptive variety; F₁, Poinsett 97×Boothbyls Blonde) were used to research the dynamic changes of the activities of membrane protective enzymes (superoxide dismutase -SOD, peroxidase-POD, catalase-CAT), the content of malondialdehyde (MDA), the content of PRO and relative conductance (RC) of the leaf under 28℃and 38℃stress. Result showed that under heat stress the increase of RC, MDA content was evidently lower; the increase of the SOD, POD activity and the content of PRO was evidently higher; the decreased of CAT activity was evidently lower in the heat tolerance variety than the heat susceptive variety. The positive heterosis of POD was observed, negative hetersis of the others index were found in F₁.3 Isolation of gene of heat response related in cucumber seedlings and study on the molecular markers linked the heat tolerance in cucumberThe method of Trizol isolation of total RNA was modified to efficiently isolate high quality total RNA from cucumber young leaves. Three clear bands of 28S, 18S and 5S could be seen clearly by agarose gel electrophoresis and no degradation was observed. The yield of isolated RNA was high and 100μg to 150μg RNA per 0.1 grams of young leaves could be harvested. The results from synthesis of double strand cDNA and cDNA-AFLP indicated that the quality and quantity of the isolated RNA was sufficient for conducting those experiments. This method can be used to isolate RNA from cucumber young leaves for cNDA-AFLP.'Xishuangbanna' and 'Erzaozi' were used the material of examination. The seedling was heat treated with high temperature (38℃) for 2h. The isolation of gene was performed with cDNA-AFLP technique. The gene expression was detected using RT-PCR technique. The chrr 182 (cucumber heat response related) which is the specific fragment of the heat response was isolated from cDNA of the heat sensitive cucumber cultivar 'Erzaozi' under heat stress. The results of blastx in GenBank of NCBI indicated that chrr182 had only homolosaus with the transcription-repair coupling factor gene. The expression of chrr182 was enhanced at 38℃for 2h induced. The cucumber seedling heat tolerance or heat injure could be related to the response of the transcription-repair coupling factor gene under heat stress.The heat tolerant cultivar 'Poinsett 97' and the heat sensitive cultivar 'Boothbyls Blonde' were used to construct F₂ population. Bulked segregant analysis was used to screen the candidate SSR markers. The screened markers were analyzed in the F₂ population; CMCT505 candidate markers, which possibly associated to the high temperature resistance, were gotten. After analyzed with F₂ population, This SSR marker was found to be associated with heat tolerance and the linkage distance was 19.3 cM. SSR molecular markers linked to heat tolerance was selected in this research. It will lay a foundation forheat tolerance line breeding in cucumber.4 Cloning, expression and structural analysis of introns of the CSHSP70 gene incucumberThe heat tolerant cultivar 'Xishuangbanna' and the heat sensitive cultivar 'Erzaozi' were used the material of examination. The seedlings were treated with 25℃,38℃and 42℃for 2h. The special primers were designed according to the cDNA sequence of the HSP70 gene of the cucumber chloroplastic stroma (CSHSP70) . The expression of CSHSP70 gene was detected using RT-PCR technique. At 25℃the expression of CSHSP70 gene were not observed in both the heat tolerant cultivar 'Xishuangbanna' and heat sensitive cultivar 'Erzaozi'. At 38℃, the CSHSP70 expression was obviously increased in 'Erzaozi' and was not observed in 'Xishuangbanna'. At 42℃, the expression of CSHSP70 gene was all obviously increased in 'Xishuangbanna' and 'Erzaozi'. The expression of CSHSP70 gene was increased under heat stress. The results are important for understanding the molecular mechanism of the cucumber heat injury and heat tolerance.Introns widely exist in the DNA regions of non-encoding proteins and it could effectively regulate gene expression in the eukaryote. The objective of this study was to examine the number and structure of the introns of the chloroplastic stroma HSP70 (CSHSP70) gene in cucumber. Primers were designed according to the cDNA sequence from cucumber CSHSP70 gene and cucumber CSHSP70 gene was cloned from gDNA using polymerase chain reaction (PCR) technique. The existence of introns was shown by reverse transcription polymerase chain reaction (RT-PCR) technique. After sequencing the three fragments cloned from cucumber CSHSP70 gene, a 4056bp cucumber CSHSP70 gene with seven introns was obtained. Analysis of these introns indicated that they are rich in A·T, have similar structure of promoter and core structure of the HSE. Comparative analysis of the CSHSP70 part sequence in cucumber, melon and luffa indicated that its nature is conservative among those of other cucurbit crops. The introns maybe be play an important role in its expression in the cucumber CSHSP70 gene.