| Konjac, a thermophilic without heat-tolerance crop, and high temperature is one of the major abiotic stress effecting its yield and quality. In southwest China mainly konjac planting area, especially in Sichuan and Chongqing, periodic thermal damage often occurs, thus screening heat resistant germplasm resources and breeding new heat resistant varieties have become particularly important. Identification of heat tolerant germplasm is the foundation of heat-tolerance breeding in konjac, however, recently we knew little about the details of heat-resistant physiological and molecular mechanism, and their interaction of regulation mechanism. This research selected three genera of konjac with obvious differences in heat-tolerance as experimental materials,studying tolerant physiological basis and regulation mechanism in konjac on two aspects of physiology and molecular biology in order to establishing foundation to further considering expression regulation and functional analysis of heat-tolerant factors in konjac.In this experiment, Amorphohallus albus, Amorphohallus konjac and Amorphohallus bulbifer were selected as materials due to their obvious differences in heat-tolerance. Through measuring the variations of index and content of physiology and biochemistry under 43℃,observing leaf microstructure,and stating the changes of heat damage index,we mastered the change law of main heat-tolerant index in 3 genera of konjac materials,determining the strong and weak consistency of heat-tolerance in different genera of konjac and selecting the index of reflecting the differences between different konjac genera effectively, discussing the heat-tolerant physiological mechanisms of konjac;Designing primers by utilizing published homologous of heat shock protein, we cloned the heat shock protein, s HSP,HSP70 and HSP90 genes of 3 genera of konjac materials,and researched expression of s HSP, HSP70 and HSP90 in leaves of konjac materials in different times of heat stress by designing semi-quantitative RT-PCR primers based on sequencing sequence. The main results are as follows: 1. Identification of heat tolerance for konjacUsing significantly different heat-resistant konjac as the material(Amorphohallus albus, Amorphohallus konjac and Amorphohallus bulbifer), heat-related indicators of different konjac leaves were measured under 43℃. The result showed that the size of CAT activity, SP and Pro content and the increase in Aa leaves was more than Ak and Ab, Ab later, Ak last;The content of REC and MDA and the increase in Ak leaves was highest, Ab later, Aa last;After exposed to 6-, 18-, or 24-h high temperature,the size of the activity of SOD, POD and the increase in Aa leaves was significantly more than Ak and Ab;After treated 6h with high temperature, the content and the increase of SS in Aa leaves was more than Ak and Ab At the same time,the size of Pn, Tr, and Chl a/b and the decrease in Aa leaves was more than Ak and Ab during high temperature stress;After treated 1-, 2-, and 4-d, the size of Gs and Ci in leaves is Aa, Ab, and Ak by size. This showed that the change rule of ten heat-tolerant indexes including CAT, SP, Pro, REC, MDA, Chl a/b, Pn, Tr, microstructure and heat injury index could reflect the difference of heat tolerance in konjac accurately,which can be used for identify heat tolerance. The strong and weak of heat tolerance in 3 genera konjac is Aa, Ab, and Ak successively. The index of POD activity varied complexly under heat stress, and there was no significant correlation among different genera. 2. Cloning and sequence analysis of HSP gene from konjacAmorphohallus albus, Amorphohallus konjac and Amorphohallus bulbifer were selected as materials,and the size of Aas HSP, Aks HSP and Abs HSP gene fragments cloned by homology cloning are 468 bp, which contains the full-length open reading frame and encodes 155 amino acid residues. Bioinformatics analysis showed that the molecular weight of Aas HSP, Aks HSP and Abs HSP protein is 17.284, 17.270 and 17.095 k Da, and p I is about 6.84, 6.84, and 6.31, respectively;C-terminal of the amino acid sequence of three genes all contain typical ACD domain;This showed the closest relationship with Pinellia Pts HSP. The size of Aa HSP70, Ak HSP70 and Ab HSP70 gene fragments contains the full-length open reading frame were 1944 bp, 1947 bp and 1944 bp, respectively, encoding 647, 648, and 647 amino acid residues. Bioinformatics analysis results showed the molecular weight of Aa HSP70, Ak HSP70 and Ab HSP70 is 71.069, 71.054 and 71.023 k Da, and p I is 5.14, 5.16 and 5.17, respectively. The N-terminal of amino acid sequence of three genes contain highly conserved NBD domain;Aa HSP70, Ak HSP70 and Ab HSP70 have the closest relationship with musa acuminate Ma HSP70 and Gossypium raimondii Gr HSP70. All of the size of Aa HSP90, Ak HSP90 and Ab HSP90 gene fragments are 2103 bp, which contains the full-length open reading frame and encodes 700 amino acid residues. Bioinformatics analysis results show the molecular weight of Aa HSP90, Ak HSP90 and Ab HSP90 is 80.036, 80.056 and 80.188 k Da, and p I is 5.01, 5.01 and 5.00, respectively. The N-terminal of amino acid sequence of three genes contains highly conserved ATPase domain. Aa HSP90, Ak HSP90 and Ab HSP90 have the closest relationship with musa acuminate Ma HSP90. 3. RT-PCR analysis of HSP gene under high temperature stressThis article slected Amorphohallus albus, Amorphohallus konjac and Amorphohallus bulbifer as materials, using the semi-quantitative RT-PCR method to the situation of expression in leaves in different time under high temperature stress. The result showed that the expression of thermal variety Aks HSP rapidly increased to the highest value after 0.5 hour under heat stress, but the expression of heat resistance variety Aas HSP and Abs HSP, respectively, reach the peak after 2 hours under heat stress. The peak of Aas HSP is largely higher than the Aks HSP and Abs HSP. In addition, response time of gene expression of Aas HSP and Abs HSP is longer than the Aks HSP. The expression of thermal variety Ak HSP70 increased to the highest value after 0.5 hour under heat stress, but the expression of heat resistance variety Aa HSP70 and Ab HSP70 reached the peak after 2 hours and 5 hours under heat stress. The peak of Aa HSP70 is largely higher than the others. Further, response time of gene expression of Aa HSP70 and Ab HSP70 is longer than the Ak HSP70. The expression of thermal variety Ak HSP90 rapidly increases to the highest value after 0.5 hour under heat stress, but the expression of heat resistance variety Aa HSP90 and Ab HSP90, respectively, reach the peak after 2 hours and 1 hour under heat stress. The peak and total expression of Aa HSP90 is obviously higher than the Ak HSP90 and Ab HSP90. Moreover, response time of gene expression of Aa HSP90 and Ab HSP90 is longer than the Ak HSP90. |
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