Function and Effect Analysis of a New Gene Wx410 Regulating Amylose Synthesis in Rice

doi:10.16819/j.1001-7216.2022.220103

Abstract

Abstract:

【Objective】 Mining new allelic variants of the Wx gene and identifying the effect of the new Wx410 allele on rice quality traits. 【Method】 Using Wx^lv,Wx^a and Wx^b alleles as templates, the A-G point mutation at base 101 of exon 10 was performed by PCR, and Wx410 sentinel mutation plant expression vectors in different Wx allelic backgrounds (pEGFC-Wx^lv410, pEGFC-Wx^a410 and pEGFC-Wx^b410) were constructed respectively, and the genetic effect of the variation at this locus on rice quality was analyzed by transforming the glutinous rice variety Suyunuo. 【Results】 The expression level of the Wx gene in the endosperm of the transgenic plants pEGFC-Wx^lv410, pEGFC-Wx^a410 and pEGFC-Wx^b410 7 and 14 days after flowering was not significantly different from that of the positive control, while the activity of GBSS Ⅰ was highly significantly lower than that of the positive control. The transgenic plants had significantly lower amylose content in mature seeds compared to the wild-type. pEGFC-Wx^a410 and pEGFC-Wx^b410 had significantly higher gelatinous consistency and pEGFC-Wx^lv410 had significantly lower gelatinous consistency of endosperm compared to the positive control. 【Conclusion】 Wx410 is a new functional allele for starch synthesis in rice. The allele explains 4%−6% of amylose content variation, which just fills the gap in the range of amylose content regulated by the currently identified complex alleles, providing a richer genetic resource for rice taste and processing-related quality improvement.

Key words: rice, Wx gene, amylose content

摘要：

【目的】挖掘Wx新等位变异，明确Wx410新等位基因对稻米品质性状的影响。【方法】以Wx^lv、Wx^a和Wx^b等位基因为模板，利用PCR进行第10外显子第101位碱基的A-G单点突变，分别构建了不同Wx等位背景下的Wx410定点突变植物表达载体pEGFC-Wx^lv410、pEGFC-Wx^a410和pEGFC-Wx^b410，阳性对照组载体分别为pEGFC-Wx^lv、pEGFC-Wx^a和pEGFC-Wx^b。通过转化糯稻品种苏御糯，分析该位点的变异对稻米品质的遗传效应。【结果】花后7 d和14 d，转基因植株pEGFC-Wx^lv410，pEGFC-Wx^a410及pEGFC-Wx^b410的胚乳Wx基因表达量较各自的阳性对照材料无显著变化，而颗粒结合淀粉合酶活性极显著降低；转基因植株直链淀粉含量较野生型显著降低，而糊化温度无明显变化；pEGFC-Wx^a410及pEGFC-Wx^b410的胶稠度较各自的阳性对照材料显著升高，而pEGFC-Wx^lv410的胶稠度较其阳性对照材料显著降低。【结论】pEGFC-Wx410为水稻淀粉合成的一个新的功能等位基因，控制的直链淀粉含量为4%~6%，刚好弥补了目前所鉴定的复等位基因所调控的直链淀粉含量在这个范围内的空缺，为稻米食味和加工相关品质改良提供更丰富的遗传资源。

关键词: 水稻, Wx基因, 直链淀粉含量

MAO Hui, PENG Yan, MAO Bigang, SHAO Ye, ZHENG Wenjie, HU Liming, ZHOU Kai, ZHAO Bingran. Function and Effect Analysis of a New Gene Wx410 Regulating Amylose Synthesis in Rice[J]. Chinese Journal OF Rice Science, 2022, 36(6): 579-585.

毛慧, 彭彦, 毛毕刚, 韶也, 郑文杰, 胡黎明, 周凯, 赵炳然. 水稻直链淀粉合成调控新基因Wx410的功能与效应分析[J]. 中国水稻科学, 2022, 36(6): 579-585.

Figures/Tables 8

Table 1. Sequence of primers used in the study.

引物名称 Primer name	正向引物序列 Forward primer sequence(5′-3′)	反向引物序列 Reverse primer sequence(5′-3′)
Wx	WxF1: GCCGGAGGGCCGTTCGACGGCA	WxR1: TACTAAAATTGGTTGGATTCTGA
	WxF2: GCCGAGTTGGTCAAAGGAA	WxR2: TCCAGCCTGCCGATGAACGCGATC
	WxF3: GGAACAGAAGGGCCCTGACG	WxR3: ATGGCATGGTATAATATGGAACAG
KASP1	F1: GAAGGTGACCAAGTTCATGCTTTCCAGGGCCTCAAGCCCC	R1: CGCTGGTCGTCACGCTGA
	F2: GAAGGTCGGAGTCAACGGATTGTTCCAGGGCCTCAAGCCCA
KASP2	F1: GAAGGTGACCAAGTTCATGCTGCGTTCATCGGCAGGCTGGA	R1: TTTGGCATATCGTGCAAGTGTGTCT
	F2: GAAGGTCGGAGTCAACGGATTGCGTTCATCGGCAGGCTGGG
qWx	qWx-F: ACCTGACACTGGAGTTGATTAC	qWx-R: GTATGGGTTGTTGTTGAGGTTTAG
qActin	qActin-F: ACCTGACACTGGAGTTGATTAC	qActin-R: GTATGGGTTGTTGTTGAGGTTTAG
qHyg	qHyg-F: GCTTCTGCGGGCGATTTGTGT	qHyg-R: GGTCGCGGAGGCTATGGATGC

Fig. 1. Schematic diagram of Wx410 targeted mutant plant expression vector under different Wx allelic backgrounds and identification of T0 transgenic plants. M, DNA marker; Lanes 1-17, Hygromycin detection of different Wx410 transgenic lines; Lanes 18-24, Hygromycin test of control material.

Fig. 2. KASP genotyping of transgenic T1 generation materials. A, Molecular marker KASP1 identifies transgenic T1 generation positive control plants (based on 23 bp nucleotide insertion in the second exon of Wx allele); B, Molecular marker KASP2 identifies homozygous plants of Wx410 targeted directed mutant transgenic line (based on SNP A-G at position 101 of exon 10 of Wx gene); Green, blue, red and gray dots represent transgenic receptors, homozygous lines, heterozygous materials and H2O, respectively.

Table 2. Comparison of agronomic traits between transgenic materials and control materials.

材料 Material	株高 Plant height /cm	有效穗数 No. of effective panicles	每穗总粒数 No. of grains per panicle	结实率 Seed setting rate/%	千粒重 1000-grain weight /g	精米长宽比 Length to width ratio of milled rice
pEGFC	132.7±0.6 a	8.7±0.8 a	82.3±4.2 a	79.6±0.8 a	36.1±0.3 a	2.0±0.1 b
pEGFC-Wx^lv	131.7±1.5 a	8.7±0.6 a	81.3±3.2 a	79.8±1.0 a	36.0±0.8 a	2.2±0.0 a
pEGFC-Wx^lv410	131.0±2.0 a	8.7±0.6 a	80.0±4.6 a	78.3±2.6 a	36.6±0.3 a	2.1±0.0 ab
pEGFC-Wx^a	132.0±1.0 a	8.7±0.8 a	82.0±3.0 a	80.4±1.9 a	36.2±0.3 a	2.2±0.0 a
pEGFC-Wx^a410	131.3±0.6 a	8.3±0.6 a	84.3±5.7 a	80.4±2.0 a	36.7±0.3 a	2.1±0.0 ab
pEGFC-Wx^b	130.9±1.6 a	8.7±0.6 a	82.0±3.5 a	78.9±2.6 a	36.4±0.3 a	2.2±0.0 a
pEGFC-Wx^b410	129.3±1.5 a	8.7±0.6 a	78.0±4.4 a	79.0±1.7 a	35.8±0.6 a	2.1±0.0 b

Fig. 3. Comparison of grain appearance of transgenic plants.

Fig. 4. Determination of relative expression level of Wx gene at different time after anthesis. A, 7 days after flowering; B, 14 days after flowering; Mean±SD, n=3. pEGFC, Negative control; pEGFC-Wxlv, pEGFC-Wxa and pEGFC-Wxb, Positive control; pEGFC-Wxlv410, pEGFC-Wxa410 and pEGFC-Wxb410, Wx410 targeted mutant transgenic lines under different Wx allelic backgrounds. The same below.

Fig. 5. OsGBSSⅠ activity in endosperm at different time after anthesis. A, Seven days after flowering; B, 14 days after flowering; Mean±SD, n=3; * and ** represent significant difference between Wx410 targeted mutant transgenic line and its positive control at 0.05 and 0.01 levels by t-test, respectively.

Fig. 6. Determination of endosperm physical and chemical properties of transgenic plants. Mean±SD, n=3; ** represent significant difference between Wx410 targeted mutant transgenic line and its positive control at 0.01 level by t-test.

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