Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (12): 2331-2339.doi: 10.3864/j.issn.0578-1752.2020.12.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Expression Analysis of the Chlorophyll Biosynthesis Structural Genes in Green and White Foxtail Millet [Setaria italica (L.) Beauv]

ZHANG Bin,LI Meng,LIU Jing,WANG JunJie,HOU SiYu,LI HongYing,HAN YuanHuai()   

  1. College of Agriculture, Shanxi Agricultural University, Taigu 030800, Shanxi
  • Received:2019-11-03 Online:2020-06-16 Published:2020-06-25
  • Contact: YuanHuai HAN E-mail:swgctd@163.com

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Abstract:

【Objective】 The phenotypic differences of millet color in foxtail millet is caused by the different kinds of pigment accumulation in grains. The phenotype of millet color, the indicators from color system, the chlorophyll contents, the ultra-structure of inner grains, and the expression profile of chlorophyll biosynthesis related genes were compared and analyzed between the green and white millet varieties. Our main objectives are to find the key gene causing the millet color difference between green and white millet, and to explore the mechanism for the formation of green millet color. 【Method】 In this study, the millet color indicators （L*, a*, b*）were determined using the color system. The Chla, Chlb, and Chl contents in grains were measured by spectrophotometer separately, and the inner ultra-structure including the size and numbers of the starch and chromoplast in the foxtail millet grains were observed and analyzed at mid stage of grouting by transmission electron microscope. Meanwhile, the expression patterns of 18 genes distributing in the upstream and downstream of the chlorophyll biosynthesis were analyzed among three varieties with different millet color by qRT-PCR. The SiCAO gene was separately cloned from three varieties with the DNA of seedling leaves as templates, and then were compared with each other. 【Result】 Hunted millet of Daqinggu and Lumiqinggu were green colors, and the a* color indicators of them were lower than Niumaobai with white millet color. The Chla, Chlb and Chlcontains in the grains of green varieties were all significantly higher than white variety, and only 0.006 mg·g-1 Chlb were detected in white variety. The results of qRT-PCR showed that the expression of SiCAO gene couldn’t be detected in white variety, but it had high expression levels in two green varieties. The significant different expression level of SiCAO was one of the important reasons leading to the color difference between two millet color millet varieties. The SiCAO gene with an open reading frame of 1626 bp was separately cloned from three varieties, and their sequences showed differences at 171, 184, 195, 286 and 318 amino acid sites. Simultaneously, ultra-structural observations indicated that the endosperm layer of the grain is mainly consisting of the single-amyloplast. Compared with the white variety, the starch in green varieties were bigger and darker, and surrounded by more chromoplasts and spherosomes, and these differences might affect the pigments accumulations in grains. 【Conclusion】 SiCAO, which is over-expressed in green varieties but not expressed in white variety, is one of the important reasons that causing different amount of chlorophyll contents accumulation leading to green and white millet colors.

Key words: foxtail millet, chlorophyll, biosynthesis, SiCAO, ultra-structure

Fig. 1

The phenotype comparison between white and green millet varieties A: Niumaobai; B: Daqinggu; C: Lumiqinggu"

Table 1

The color indicators of different foxtail millet varieties"

小米品种Millet varieties 亮度值L* 黄/蓝度值a* 红/绿度值b*
NMB 66.91±0.12a 4.99±0.10a 28.95±0.26a
DQG 43.92±0.50b 2.66±0.14b 22.97±0.29c
LMQG 41.10±0.18c 2.93±0.07b 25.31±0.48b

Fig. 2

The comparison of chlorophyll contents in different millet color varieties NMB: Niumaobai; DQG: Daqinggu; LMQG: Lumiqinggu. Different letters indicate a statistically significant difference (Dunnett’s test, P<0.05). The same as below"

Fig. 3

Quantitative real-time RT-PCR analysis of the expression profiling of the chlorophyll biosynthetic genes in white cultivate NMB and green cultivates DQG and LMQG"

Fig. 4

Protein sequence alignment of SiCAO in NMB, DQG and LMQG"

Fig. 5

Ultrastructure of white and green millet grains A: Grain of white millet; B: Grain of green millet. S: Starch"

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