‘黑比诺’葡萄不同叶龄叶片叶绿体内淀粉积累及其相关基因表达差异分析

doi:10.3864/j.issn.0578-1752.2022.21.013

Abstract

Abstract:

【Objective】The accumulation of starch in chloroplast of grape leaves increased with leaf maturation, but the related molecular mechanism has not been reported. In this study, the potential related genes were screened in order to clarify the molecular regulation mechanism of starch accumulation in chloroplast of grape leaves with different leaf ages.【Method】 Two years old ‘Pinot Noir’ grapes (Vitis vinifera L.) were used as plant materials to investigate the dynamic changes of starch accumulation in chloroplast of leaves with different ages. The RNA sequencing technology was used to characterize the differently expressed genes (DEGs) involved in starch and sucrose metabolism in leaves with different ages. The expression levels of several key candidate genes were detected by using real-time quantitative qPCR.【Result】The starch grains in NL (undeveloped leaves) were smallest in volume and lowest in number compared with these in GL (growing leaves) and ML (mature leaves). With the increase of leaf ages, the volume and amount of starch grains in GL and ML also increased. A total of 58.57 GB valid data was obtained by RNA-sequencing. Differentially expressed genes mainly involved in starch and sucrose metabolism pathway for the leaves with different ages. The genes involved in the process of sucrose conversion into starch synthesis precursor adenosine diphosphate glucose (ADP) showed up-regulated expression level with the increase of leaf ages, including cell wall invertase (CWINV), phosphoglucomutase (PGM), and ADP-glucose pyrophosphorylase (AGPase). The genes involved in starch synthesis and hydrolysis also presented up-regulated expression level with the increase of leaf ages, including soluble starch synthase (SSⅠ), starch branching enzyme (SBE), α-amylase (AMY), and β-amylase (BAM). AGPase, SSⅠ and SBE played an important role in synthesis of amylopectin with semi-crystalline structure.【Conclusion】With the increase of leaf ages, the starch accumulation in chloroplast also increased. AGPase, SSⅠ and SBE genes could be the key genes involved in the regulation of starch accumulation in chloroplast of grape leaves.

Key words: Pinot Noir, transcriptome, leaf age, starch, starch metabolism

YOU JiaLing,LI YouMei,SUN MengHao,XIE ZhaoSen. Analysis Reveals the Differential Expression of Genes Related to Starch Accumulation in Chloroplast of Leaf with Different Ages in Pinot Noir Grape[J].Scientia Agricultura Sinica, 2022, 55(21): 4265-4278.

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基因名称 Name	基因ID Gene ID	正向引物 Forward primer sequence (5′-3′)	反向引物 Reverse primer sequence (5′-3′)
VvActin7	VIT_04s0044g00580	F: CTTGCATCCCTCAGCACCTT	R: TCCTGTGGACAATGGATGGA
VvCWINV1	VIT_09s0002g02320	F: TTGACAACGCTAAGAACCGAAGAA	R: GAATTGCCTGAACTCCAGACCATC
VvHXK2	VIT_09s0002g03390	F: GAGCACTACAGTGAGTACAGTAAG	R: GAATGAGAGGCAGCAAGAAGG
VvFRK1	VIT_15s0048g01260	F: AAGTCTGGTAGCATCCTCTCTTAT	R: TCAGCCTGGTTCCATATACTCAT
VvFRK2	VIT_05s0102g00710	F: GCTGTAGAGGGATTTCATGTCAA	R: CTCTCAATCTTGCCTCGTCTTC
VvFRK4	VIT_18s0089g01230	F: GCATCAAGAGCATCTGGAACC	R: ACCATCAGTGACCACAAGCA
VvPGM	VIT_01s0011g05370	F: GCAACCATCCGTGTCTATATTGA	R: TTGAGAGCAACCTCCACAAGA
VvAGPS1.2	VIT_03s0038g04570	F: GTGCCATTCTATGCCATACTGAT	R: GAATCCGCTACTGGTGGTGTA
VvSBEⅠ	VIT_08s0007g03750	F: GGAGACAGAATAACCAAGATGACA	R: GCTGGCAAGATGATACCTTACAA
VvSBEⅡ	VIT_18s0001g00060	F: GCTGAGCAGGAGAGTCTTGA	R: GTAGCCTCTTCCACCACATCT
VvAMY1.1	VIT_03s0063g00400	F: CAACCTTAACGGCTTCTCCAAT	R: ATGAGTGATTCCAGAGGCAGATA
VvAMY1.2	VIT_03s0063g00450	F: AGCAAGACGCAAGAGGAATATG	R: GCACTGTAGTCATCTCCAGAATC
VvAMY2	VIT_18s0001g00560	F: GCCACCAGCATCTCAGTCT	R: ACTATGTCAGCCATTGCTCTAAC
VvBAM2	VIT_15s0046g02620	F: TTGTGATGGAGGCAAGTATGATAG	R: TGGCTAGAGCAAGTACACGAT
VvBAM3	VIT_02s0012g00170	F: GGCTGAATTAACTGCTGGATACTA	R: TTGCTGCTCCCTGTCCTTC
VvSSⅠ	VIT_16s0098g01780	F: TAGTGCCAGTGCTTTTGGCT	R: CACAGCCCCATACCACTCAG

样品名称 Sample name	质控后的 clean reads数 Total reads	比对到参考基因组上的reads数及百分比 Total map reads (%)	比对到唯一位置的reads数及百分比 Unique map reads (%)	比对到多个位置的reads数及百分比 Multi map reads (%)	比对到参考基因组正链上的reads数及百分比 Positive map reads (%)	比对到参考基因组负链上的reads数及百分比 Negative map reads (%)
NL1	43198062	39524089 (91.5%)	38617667 (89.4%)	906422 (2.1%)	19289169 (44.65%)	19328498 (44.74%)
NL2	42652276	39262139 (92.05%)	38352259 (89.92%)	909880 (2.13%)	19157641 (44.92%)	19194618 (45.0%)
NL3	41550992	38222716 (91.99%)	37345353 (89.88%)	877363 (2.11%)	18653976 (44.89%)	18691377 (44.98%)
GL1	43373766	39810586 (91.78%)	38866791 (89.61%)	943795 (2.18%)	19454902 (44.85%)	19411889 (44.75%)
GL2	45964018	42408116 (92.26%)	41440537 (90.16%)	967579 (2.11%)	20735166 (45.11%)	20705371 (45.05%)
GL3	42560230	39115832 (91.91%)	38227440 (89.82%)	888392 (2.09%)	19122008 (44.93%)	19105432 (44.89%)
ML1	44057236	40379107 (91.65%)	39408268 (89.45%)	970839 (2.2%)	19690278 (44.69%)	19717990 (44.76%)
ML2	44139002	40538462 (91.84%)	39625981 (89.78%)	912481 (2.07%)	19802759 (44.86%)	19823222 (44.91%)
ML3	43029030	39519670 (91.84%)	38610003 (89.73%)	909667 (2.11%)	19297553 (44.85%)	19312450 (44.88%)

基因ID Gene ID	基因名称 Gene name	FPKM			GL vs NL		ML vs GL		ML vs NL
基因ID Gene ID	基因名称 Gene name	NL	GL	ML	Log2FC	P_adj	Log2FC	P_adj	Log2FC	P_adj
VIT_09s0002g02320	CWINV	9.46	28.66	32.22	1.59	2.91E-09	0.13	6.37E-01	1.77	1.12E-16
VIT_09s0002g03390	HXK2	1.19	0.54	0.00	-1.13	3.32E-02	-6.25	2.60E-05	-7.33	4.42E-08
VIT_15s0048g01260	FRK1	5.74	3.21	0.58	-0.84	7.46E-03	-2.52	1.18E-08	-3.32	3.60E-17
VIT_05s0102g00710	FRK2	79.24	57.48	21.44	-0.47	1.14E-04	-1.46	1.60E-31	-1.88	6.07E-43
VIT_18s0089g01230	FRK4	7.45	9.78	0.15	0.39	3.40E-01	-6.10	1.32E-25	-5.67	8.66E-24
VIT_01s0011g05370	PGM	59.83	68.55	149.43	0.19	2.68E-01	1.09	6.58E-16	1.32	4.67E-36
VIT_03s0038g04570	AGPS1.2	1.63	1.97	50.92	0.27	4.68E-01	4.66	1.56E-17	4.97	1.15E-17
VIT_16s0098g01780	SSⅠ	12.37	64.56	151.46	2.38	6.35E-86	1.19	1.79E-41	3.62	6.82E-223
VIT_08s0007g03750	SBEⅠ	10.74	18.68	75.62	0.79	1.67E-05	1.98	4.39E-91	2.82	5.34E-63
VIT_18s0001g00060	SBEⅡ	46.07	53.83	96.15	0.22	1.17E-01	0.80	7.37E-12	1.06	1.02E-35
VIT_03s0063g00400	AMY1.1	0.95	5.34	10.63	2.49	6.30E-10	0.95	1.26E-03	3.49	1.92E-41
VIT_03s0063g00450	AMY1.2	0.27	1.21	1.09	2.15	3.67E-03	-0.19	8.04E-01	2.00	2.26E-02
VIT_18s0001g00560	AMY2	5.33	9.79	19.47	0.87	3.78E-07	0.95	5.26E-11	1.87	7.50E-33
VIT_15s0046g02620	BAM2	10.80	17.79	33.68	0.71	2.63E-07	0.88	1.50E-14	1.64	2.38E-42
VIT_02s0012g00170	BAM3	1.19	26.15	93.39	4.45	7.37E-38	1.80	2.47E-14	6.29	1.27E-147

Analysis Reveals the Differential Expression of Genes Related to Starch Accumulation in Chloroplast of Leaf with Different Ages in Pinot Noir Grape

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