异源过表达OsATG8b基因提高转基因拟南芥的 氮/碳胁迫耐受性和产量

doi:10.11983/CBB18064

摘要/Abstract

摘要： 氮素是参与植物生长发育的一种重要元素, 对植物的产量和品质具有重要作用。自噬是真核生物中一种保守的细胞组分降解-循环再利用途径, 在植物生长发育和籽粒形成期间的氮素再动员过程中发挥作用。我们鉴定到水稻(Oryza sativa)自噬核心基因OsATG8b, 并获得2个独立的35S-OsATG8b转基因拟南芥(Arabidopsis thaliana)纯合株系。研究表明OsATG8b基因响应低氮胁迫处理, 过表达OsATG8b基因促进转基因拟南芥的生长发育, 使莲座叶增大, 单株产量显著提高(15.16%)。进一步研究表明, 过表达OsATG8b能够显著增强缺氮胁迫下转基因拟南芥叶片中的自噬活性, 从而有效缓解氮胁迫和碳胁迫对转基因拟南芥造成的生长抑制。因此, OsATG8b是提高氮素利用效率和产量的候选基因。

关键词: 自噬, OsATG8b, 氮素再利用, 产量

Abstract: Nitrogen is an essential element for plant growth and development and plays an important role in plant yield and quality. Autophagy is a conserved degradation-recycle pathway of cellular components in eukaryotes that plays an important role in nitrogen remobilization during plant growth and grain formation. We identified an autophagy core gene OsATG8b in rice and obtained 2 independent 35S-OsATG8b transgenic Arabidopsis homozygous lines. The expression of OsATG8b responded to nitrogen starvation in rice. Overexpression of OsATG8b promoted the growth and development of transgenic Arabidopsis, with rosette leaves larger than wild-type leaves. In addition, the yield increased significantly, by 15.16%. In addition, overexpression of OsATG8b could significantly enhance autophagic activity in leaves of transgenic Arabidopsis under nitrogen deficiency and effectively alleviate the growth inhibition of transgenic Arabidopsis caused by nitrogen and carbon stress. OsATG8b may be a good candidate gene for increasing nitrogen use efficiency and yield.

Key words: autophagy, OsATG8b, nitrogen remobilization, yield

甄晓溪,刘浩然,李鑫,徐凡,张文忠. 异源过表达OsATG8b基因提高转基因拟南芥的氮/碳胁迫耐受性和产量. 植物学报, 2019, 54(1): 23-36.

Xiaoxi Zhen,Haoran Liu,Xin Li,Fan Xu,Wenzhong Zhang. Heterologous Overexpression of Autophagy-related Gene OsATG8b from Rice Confers Tolerance to Nitrogen/Carbon Starvation and Increases Yield in Arabidopsis. Chinese Bulletin of Botany, 2019, 54(1): 23-36.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18064

https://www.chinbullbotany.com/CN/Y2019/V54/I1/23

图/表 9

表1 引物信息

Table 1 The information of primers

Primer name	Sequence (5′-3′)	Function
cOsATG8b-F	CCATTCAAGTGGATGGCCAAGAGCTCGTTCAAGC	Gene cloning
cOsATG8b-R	GGTGACCTAGAGCAGCCCAAAGGTGTTCTCG	Gene cloning
cpOsATG8b-F	AAGCTTAAAATTAAATAAGACGAACAGTCAAACG	Gene cloning
cpOsATG8b-R	CCATGGCGCTCCTTCCTGCACACAAT	Gene cloning
rtOsATG8b-F	GCTGATCTTACCGTTGGGCA	Real-time RT-PCR
rtOsATG8b-R	ATCAGAGCAGCTGTTGGTGG	Real-time RT-PCR
rtAtAMT1-F	GCCTCTGCTGACTACTCCAACTT	Real-time RT-PCR
rtAtAMT1-R	GACCAGAACCAGTGAGAGACGA	Real-time RT-PCR
rtAtNR1-F	AGGATGGGCTAGTAAGCATAAGG	Real-time RT-PCR
rtAtNR1-R	GCAAACTGAATCATAGGCGGTG	Real-time RT-PCR
rtAtGS2-F	CACCAAACCTTACTCTCTGACA	Real-time RT-PCR
rtAtGS2-R	CACTATCTTCACCAGGTGCTTG	Real-time RT-PCR
rtAtGDH1-F	GCTTTAGCAGCAACAAACAGAA	Real-time RT-PCR
rtAtGDH1-R	TGAGCCAATGCGTTCACTTC	Real-time RT-PCR
rtACTIN1-F	ACCATTGGTGCTGAGCGTTT	Real-time RT-PCR
rtACTIN1-R	CGCAGCTTCCATTCCTATGAA	Real-time RT-PCR
rtTIP41-F	GTATGAAGATGAACTGGCTGACAAT	Real-time RT-PCR
rtTIP41-R	ATCAACTCTCAGCCAAAATCGCAAG	Real-time RT-PCR

图1 氮胁迫处理诱导水稻幼苗叶片及根中OsATG8b基因的表达 (A) 全氮条件下生长14天的水稻幼苗转移至全氮(NS)、低氮(NL)和缺氮(ND)条件下生长1和3天时叶片中OsATG8b基因的表达量; (B) 全氮、低氮和缺氮条件下生长1和3天的水稻根系中OsATG8b基因的表达量。图中数据为平均值±标准差, n=16, 实验经3次生物学重复。**表示全氮条件与低氮和缺氮条件相比差异显著(P<0.01) (Student’s t-test)。

Figure 1 Identification of OsATG8b as a nitrogen deficiency inducible/responsive gene in leaves and roots of rice seedlings (A) The rice seedlings cultured with N-sufficient (NS) solution for 14 days and transferred to the same NS solution, low N (NL) solution and the N-deficient (ND) solution, the expression of OsATG8b gene in leaves after 1 day and 3 days treatment; (B) The expression of OsATG8b gene in roots after 1 day and 3 days treatment. Values are means±SD, n=16, three biological replicates were performed. ** indicate significant differences in NS solution compared with NL and ND solution (P<0.01) (Student’s t-test).

表2 野生型和35S-OsATG8b转基因拟南芥的抽薹及开花时间

Table 2 Bolting and flowering times of the wild-type and 35S-OsATG8b transgenic Arabidopsis

	WT	L-13	L-14
Bloting time (d)	36.56±1.58	30.78±2.07**	31.39±1.91**
Flowering time (d)	42.67±1.75	35.94±1.98**	36.61±1.94**

表3 野生型和35S-OsATG8b转基因拟南芥的产量性状

Table 3 Yield related characteristics of the wild-type and 35S-OsATG8b transgenic Arabidopsis

	Total number of siliques	Yield per plant (mg)	Thousand grain weight (mg)
WT	35.74±3.86	85.34±7.89	14.87±0.23
L-13	46.26±3.13**	100.13±6.02**	16.36±0.21**
L-14	48.22±3.62**	99.77±5.76**	17.54±0.41**

图2 过表达OsATG8b促进转基因拟南芥生长发育将苗龄8天的野生型(WT)和35S-OsATG8b转基因拟南芥幼苗移入蛭石-营养土(1:3, v/v)中。(A) 苗龄14天的35S-OsATG8b转基因拟南芥和WT幼苗中OsATG8b的表达水平; (B) 由上至下依次为移苗后10、17和24天的拟南芥表型; (C) 移苗后42天的表型; (D) 不同苗龄的35S-OsATG8b转基因拟南芥和WT的最大莲座叶半径; (E) 株高; (F) 总叶绿素含量; (G) 可溶性蛋白含量。Days: 萌发后天数。图中数据为平均值±标准差, n=24, * P<0.05, ** P<0.01 (Student’s t-test), 实验经3次生物学重复。Bars=1 cm

Figure 2 Over-expression of OsATG8b promotes growth and development of transgenic Arabidopsis 8-day-old seedlings were transferred to vermiculite-nutritional soil (1:3, v/v). (A) Expression level of OsATG8b in 14-day-old seedlings of 35S-OsATG8b transgenic lines and wild type (WT); (B) Panels from top to bottom show phenotypic observations of transgenic lines and WT of Arabidopsis at 10, 17 and 24 days after transfer to soil, respectively; (C) Phenotype of transgenic lines and WT at 42 days after transfer to soil; (D) The maximum rosette radius of 35S-OsATG8b transgenic lines and WT at different seedling age; (E) The plant height; (F) The total chlorophyll content; (G) The soluble protein content. Days: Days after germination. Values are means±SD, n=24, * P<0.05, ** P<0.01 (Student’s t-test), three biological replicates were performed. Bars=1 cm

图3 过表达OsATG8b促进转基因拟南芥抵抗缺氮胁迫 (A) 将苗龄7天的35S-OsATG8b转基因和野生型(WT)拟南芥幼苗分别移入全氮(NS)和缺氮(ND)1/2MS培养基中培养9天后的表型; (B)-(D) 分别为全氮(NS)和缺氮(ND)处理9天的35S-OsATG8b转基因和WT拟南芥幼苗的鲜重, 叶绿素和可溶性蛋白含量; (E) 苗龄7天的35S-OsATG8b转基因和WT拟南芥幼苗全氮(NS)和缺氮(ND)垂直培养9天后的表型; (F)-(H) 分别为全氮(NS)和缺氮(ND)处理9天的35S-OsATG8b转基因和WT拟南芥幼苗的主根长、地上部和地下部鲜重; (I), (J) 分别为苗龄10天的ProOsATG8b-GUS转基因拟南芥经缺氮处理和10 μmol·L-1水杨酸(SA)处理24小时后的GUS组织化学染色结果。Mock代表未经处理的拟南芥。数据为平均值±标准差, n=16, *和**分别表示各转基因株系与WT之间差异显著(P<0.05)和极显著(P<0.01) (Student’s t-test), 实验经3次生物学重复。Bars=5 mm

Figure 3 Overexpression of OsATG8b enhances tolerance to N deficiency in transgenic Arabidopsis (A) 7-day-old seedlings of 35S-OsATG8b transgenic lines and wild type (WT) were transferred to 1/2MS medium for horizontal culture with sufficient (NS) or deficient (ND) N for 9 days. (B)-(D) The fresh weight, chlorophyll content and soluble protein content in rosette leaves of WT and 35S-OsATG8b transgenic Arabidopsis under NS or ND for 9 days, respectively; (E) The phenotype of 7-day-old seedlings of 35S-OsATG8b transgenic lines and WT were transferred to vertical plates with NS or ND for 9 days; (F)-(H) The primary root length, the shoot weight and the root weight of WT and transgenic Arabidopsis lines under NS or ND for 9 days, respectively; (I), (J) 10-day-old seedlings of ProOsATG8b-GUS transgenic Arabidopsis were transferred to ND and 10 μmol·L-1 SA for 24 h, respectively. Mock represented that the seedlings without treated. Values are means±SD, n=16, * and ** indicate significant (P<0.05) and extremely significant (P<0.01) differences between transgenic lines and WT (Student’s t-test), three biological replicates were performed. Bars=5 mm

图4 过表达OsATG8b促进转基因拟南芥抵抗黑暗诱导的碳胁迫 (A) 对苗龄7天的35S-OsATG8b转基因拟南芥和野生型(WT)黑暗处理10天后记录表型(左为处理前, 右为处理后); (B) 叶绿素含量测定。数据为平均值±标准差, n=10, *表示各转基因株系与WT之间差异显著(P<0.05) (Student’s t-test), 实验经3次生物学重复。Bars=5 mm

Figure 4 Overexpression of OsATG8b in Arabidopsis enhanced tolerance to carbon starvation induced by dark treatment (A) 7-day-old seedlings of 35S-OsATG8b transgenic lines and wild type (WT) were transferred to darkness for 10 days (The left is before treatment, and the right is after treatment); (B) The chlorophyll content determination. Values are means±SD, n=10, * indicate significant difference between transgenic lines and WT (P<0.05) (Student’s t-test), three biological replicates were performed. Bars=5 mm

图5 缺氮条件下过表达OsATG8b转基因拟南芥中的自噬活性增加将苗龄7天的35S-OsATG8b转基因(L-14)和野生型(WT)拟南芥幼苗转移至含有1 μmol·L-1 ConA的缺氮培养液中处理12小时后进行MDC染色, 于激光共聚焦显微镜下观察自噬体荧光。Bars=10 μm

Figure 5 Overexpression of OsATG8b in Arabidopsis enhanced the autophagic activity under N deficient condition 7-day-old seedlings of transgenic line (L-14) and wild type (WT) were transferred to in N-deficient (ND) liquid medium with 1 μmol·L-1 ConA for 12 h, MDC-stained autophagosomes in leaves were observed by confocal microscopy. Bars=10 μm

图6 过表达OsATG8b转基因拟南芥中氮代谢关键基因的表达变化将7天苗龄的35S-OsATG8b转基因和野生型(WT)拟南芥移至全氮(NS)和缺氮(ND) 1/2MS培养基处理14天。(A) 35S-OsATG8b转基因和WT拟南芥中地上部氮代谢相关基因相对表达水平; (B) 地下部氮代谢相关基因相对表达水平。数据为平均值±标准差, n=10, *和**分别表示各转基因株系与WT之间差异显著(P<0.05)和极显著(P<0.01) (Student’s t-test), 实验经3次生物学重复。

Figure 6 Overexpression of OsATG8b in Arabidopsis changes the expression of genes in nitrogen metabolic 7-day-old seedlings of transgenic lines and wild type (WT) were transferred to 1/2MS medium with sufficient (NS) or deficient (ND) nitrogen for 14 days. (A) The expression of genes related to nitrogen metabolic in rosette leaves of 35S-OsATG8b transgenic lines and WT; (B) The expression of genes related to nitrogen metabolic in roots of 35S-OsATG8b transgenic lines and WT. Values are means±SD, n=10, * and ** indicate significant (P<0.05) and extremely significant (P<0.01) differences between transgenic lines and WT (Student’s t-test), respectively, three biological replicates were performed.

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