基于转录组测序筛选新疆野苹果组培苗应答冻害光合特性相关基因

Screening of freezing stress-responsive genes related to photosynthesis in in vitro seedlings of Malus sieversii via RNA-seq

【目的】探究新疆野苹果组培苗光合特性对冻害胁迫的响应,筛选光合特性相关应答基因。【方法】以单株系的新疆野苹果组培苗为材料,在-3℃模拟冻害条件下,观察及检测对照和0、3、6、12、24 h处理的新疆野苹果组培苗的形态、叶绿素含量、光系统Ⅱ的最大量子产量(F_(v)/F_(m))参数和以吸收光能为基础的光系统Ⅱ的性能指数(PIabs)的变化,并对前5个处理组培苗叶片进行转录组测序分析。【结果】-3℃模拟冻害胁迫下新疆野苹果组培苗叶片在12 h时开始卷缩,处理24 h时叶绿素含量显著下降,叶绿素a/b显著上升;不同处理时间点Fv/Fm与PIabs的值均显著低于对照,通过转录组测序筛选到58个参与光合调控的差异基因,其中响应光系统Ⅱ的基因有18个,响应光系统Ⅰ的基因有12个,光合电子传递链相关基因有13个,ATP合成酶相关基因有3个,细胞色素b6f相关基因有12个。同时得到PsbQ(Msi_13A005910)与PsbY(BGI_novel_G011171)基因并在冻害胁迫中极显著上调表达。根据富集结果选取7个光合相关差异表达基因,经qRT-PCR验证,基因表达趋势与转录组测序结果一致。【结论】新疆野苹果组培苗在-3℃模拟冻害条件下,F_(v)/F_(m)与PIabs均显著下降,通过转录组测序筛选到58个光合调控相关显著差异表达基因,其中PsbQ与PsbY发生极显著上调表达。PsbQ与PsbY可能会通过光氧化与光保护缓解冻害对组培苗叶绿体及光合作用产生影响。

【Objective】In order to explore the response of photosynthesis of Malus sieversii seedlings to freezing stress, we screened the responsive genes related to photosynthesis through transcriptomic analysis.【Methods】In vitro seedlings of a single plant line were used as materials. Under simulated freezing stress at-3 ℃, the changes in morphology, chlorophyll content, PSII maximum photochemical efficiency(F_(v)/F_(m)) and the performance index(PIabs) of photosystem Ⅱ of the in vitro seedlings of M.sieversii exposed to the freezing stress for 0, 3, 6, 12 and 24 h(seedlings under 25 ℃ as control) were analyzed, and the RNA-seq was performed on the leaves of the seedlings under the above five treatments.【Results】Under the freezing stress, the edge and top of the leaves began to shrink at 12 h. Compared with CK, the content of chlorophyll a decreased significantly by 16.2% within 24 h, and had no significant difference among the other four time points. Compared with CK, the content of chlorophyll b increased significantly by 52% at 0 h, but did not change significantly at 3 h, while at 6 h, 12 h and 24 h,it decreased significantly by 12.3%, 28.6% and 36.7% respectively. The change trend of total chlorophyll content was completely consistent with chlorophyll b. Carotenoid content was significantly lower than that of the control, and decreased by 23.5%, 23.5%, 25.5%, 19.6% and 29.4% at 0, 3, 6, 12 and 24 h,respectively. Compared with CK, chlorophyll a/b ratio decreased significantly at 0 h and 3 h but increased significantly at 12 h and 24 h by 14.9% and 14.1%, respectively. Under simulated freezing stress,the fluorescence images of the leaves gradually changed from dark blue to light blue and to blue-green from CK to 24 h treatment, indicating that the photochemical efficiency gradually decreased. F_(v)/F_(m)decreased significantly by 7.9%, 12.3%, 12.8%, 19.9% and 29.4% at 0, 3, 6, 12 and 24 h, respectively.Compared with CK, the PIabs decreased significantly by 61.2%, 63.7%, 78.3%, 77.2% and 87.7% at 0,3, 6, 12 and 24 h, respectively. RNA-seq analysis of samples of CK and 0, 3, 6 and 12 h, showed that total clean reads of each sample ranged from 42.48 M to 43.31 M, accounting for 95.33% to 97.35% of the total clean reads. The total clean bases for each sample averaged between 6.37 Gb-6.50 Gb, of which the number of bases with a quality greater than 30 in total clean reads accounted for 90.97%-91.67%, and the total mapping was between 86.75%-87.50%. Through KEGG enrichment analysis with a critical Q-value of ≤ 0.05, 58 genes related to photosynthesis were screened out, including 18 genes related to photosystem Ⅱ, mainly involved in oxygen evolution enhancer proteins Psb27, PsbQ, PsbY,etc. There were 12 genes involved in photosystem Ⅰ, including photosystem Ⅰ subunits PsaG, PsaN and PsaK genes. There were 13 genes related to photosynthetic electron transfer chain, including the genes of photosystem Ⅰ receptor-side ferredoxin petE, petH and petF. Three ATP synthase-related genes were atpF and atpG genes, and 12 cytochrome b6f-related genes(mainly petC genes). The expression of PsbQ(Msi_13A005910) and PsbY(BGI_novel_G011171) was significantly up-regulated by freezing stress.【Conclusion】Compared with CK, Fv/Fmand PIabs of the in vitro seedlings of M. sieversii were significantly decreased under-3 ℃. Leaves began to shrink at 12 h of freezing exposure, and chlorophylls were decreased and chlorophyll a/b ratio increased at 24 h of treatment. 58 significantly differentially expressed genes related to photosynthetic regulation were screened by RNA-seq analysis,and PsbQ and PsbY were found to be significantly up-regulated. The results provide a reference for indepth study on the molecular mechanism of the response of M. sieversii to freezing stress.