| With the increasing global warming,heat stress caused by high temperature has gained extensive attention of agricultural researchers.Heat stress affects all physiological processes of plant growth and development,leading to plant morphological and physiological changes,hindering plant development process,and ultimately leading to huge yield losses.Non-heading Chinese cabbage originated in the Taihu area of the lower reaches of the Yangtze River in China.Under different geographical distribution conditions in China,its character variations are extremely diverse.After long-term natural and artificial directional selection,a number of local varieties with different morphological and traits were finally formed,and now it has become a popular vegetable in the whole country.However,it prefers to be cold,grows best at the average temperature of 18-20?,grows weakly under the high temperature environment,and its yield and edible quality are significantly reduced.Therefore,in order to meet the needs of annual production of non-heading Chinese cabbage,how to improve its heat tolerance to achieve normal production in high temperature environment has become the primary issue of genetic breeding of non-heading Chinese cabbage.Understanding the molecular physiological response mechanism of non-heading Chinese cabbage under heat stress will help us further carry out the innovation of non-heading Chinese cabbage heat-tolerant germplasm and variety breeding.In view of this,the molecular and physiological response mechanisms of non-heading Chinese cabbage under heat stress were systematically studied,and the lipid signal gene PLD,which closely related to heat stress response was identified.The main results are as follows:1.Under heat stress,chlorophyll content and photosynthate content such as soluble sugar,soluble protein and starch in leaves of non-heading Chinese cabbage decreased,while osmotic regulators such as proline,free amino acids,superoxide dismutase,ascorbic acid oxidase and other protective enzymes increased.Heat stress has a great influence on chloroplasts in leaf cells.After heat treatment,the heat sensitive materials exhibit changes such as chloroplast deformation,lamellar thickening,osmiophage increase,and some cells even exhibit plasmolysis.After 5 d heat stress treatment,there is no obvious plasmolysis in leaf cells of heat-resistant materials,but there are also changes such as lamellar thickening and osmiophagous granules increase in chloroplasts.The results showed that heat stress had significant effects on Photosynthetic system,photosynthate,osmotic regulators,antioxidant system and cell structure of non-heading Chinese cabbage.2.RNA-Seq was used to synthetically analyze the non-heading Chinese cabbage heat-resistant material 'Gaohuaqing'.About 64.29 million reads were obtained.32,666 unigenes and 11,024 SSRs were assembled from the original sequence.A total of 1,220 differentially expressed genes were detected in heat stress treatment and control,of which 699 were up-regulated and 521 were down-regulated.Enrichment analysis showed that 12 GO subclasses and 9 KEGG pathways were significantly enriched.Finally,six differentially expressed genes were screened by qRT-PCR,these differentially expressed genes were up-regulated in varying degrees under heat stress,which was consistent with the results of RNA-Seq.3.The basic information of PLDs gene family,amino acid characteristics,chromosome location,gene structure and protein phylogenetic tree analysis,and expression pattern analysis under heat stress were carried out by bioinformatics.The results showed that 18 PLDs genes were identified in Brassica rapa and distributed in 4 subgroups,including 6 in a group and 6 in ?/? group,4 in ? group and 2 in ? group.PLDs in Brassica rapa showed inhomogeneous distribution on chromosome 3,but not on chromosome 4 and 7.The expression of 16 BrPLD genes changed after heat stress treatment.It provides a reference for further revealing the functional mechanism of PLDs gene family in Brassica rapa.4.BcPLDy was cloned by RACE technology,and its bioinformatics analysis was carried out.The expression pattern of BcPLDy in non-heading Chinese cabbage leaves under heat stress was studied by qRT-PCR.The results show that,the full length of BcPLDy gene was 2,736 bp,and the length of open reading frame was 1,905 bp,encoding 634 amino acids.The protein has no signal peptide sequence and predicts a relative molecular weight of 71.59 kDa and a theoretical isoelectric point of 6.73.The secondary structure of BcPLDy protein is mainly composed of four forms:?-helix,extended straight chain,?-rotation and irregular curl.The protein contains a plant PLD-specific C2 domain and a PLDc domain.Phylogenetic analysis showed that BcPLDy protein had the closest evolutionary relationship with Brassica napus.Under heat stress,BcPLDy expression was significantly up-regulated in three Non-heading cabbage materials with different heat tolerance,which further proved the heat tolerance correlation of BcPLDy. |
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