The latest article by Chinese scholar Nature determined the genetic mechanism of modern corn protein decline

On November 17, 2022, Beijing time, the research team of Wu Yongrui of the Center for Excellence in Molecular Plant Science of the Chinese Academy of Sciences and the research team of Wang Wenqin of Shanghai Normal University published a research paper
entitled "THP9 enhances seed protein content and nitrogen-use efficiency in maize" in Nature 。 After 10 years of unremitting efforts, the researchers finally cloned Teosinte High Protein 9 (THP9),
a key mutant gene that controls the formation of high protein quality and efficient nitrogen utilization in maize from wild maize.

The ancestor of corn originated in the Balsas River basin in southern Mexico in South America, called Bulbophyllum grandiflora, which grew like a weed and the seeds were wrapped in a hard shell that could not be eaten
directly.
Human ancestors began to domesticate corn as early as 9,000 years ago, and gradually transformed the weed-like wild corn grass into today's corn.

Today, corn is one of the world's most productive crops, with an annual global output of 1.
2 billion tons, and China produces 270 million tons a year
.
Among them, 70% of corn is used as feed, corn yield is high, effective energy is more, is the most commonly used and the largest amount of feed, so it has the reputation of "feed king
".
With the improvement of people's quality of life, the demand for meat, eggs and milk is increasing, and the consumption of corn is also increasing, resulting in the continuous increase
of corn imports in recent years.
Due to the low protein content of ordinary corn grains, most hybrid seeds have less than 8% of grain protein, so soybean protein needs to be supplemented in feed, but soybeans are heavily dependent on imports, which has become the "stuck neck" problem of China's livestock and poultry breeding industry
.
If the content of ordinary corn protein increases by one percentage point, it is equivalent to China can import nearly 8 million tons of soybeans! Therefore, increasing the content of corn protein is not only a major strategic need to ensure national food security, but also one of the important ways to ensure the healthy development of
China's livestock and poultry breeding and feed processing industry.
However, the mechanism of the formation of high protein in wild maize has been a long-standing problem of the century, and the key genes to control the total protein content and efficient use of nitrogen in maize have not been found
.

In 2012, the research team began to conduct the search for corn high-protein donor materials, protein content determination, genetic analysis, and population construction
.
Experiments found that the inbred protein content of ordinary maize was about 10%, while the seed protein content of wild maize was as high as 30% without nitrogen fertilizer, which was 3 times that of modern common cultivated maize, indicating that wild maize contained key genes
to control high protein content.
What are these genes, and what exactly have they changed in wild and modern corn? Can they be mined to increase the protein content of modern corn? The genetic variation of different corn inbred lines is greater than the difference between humans and chimpanzees, and the difference between wild maize 9,000 years ago and modern maize is even
greater.

In order to make full use of the genetic resources of wild maize and mine the excellent mutant genes that control its high protein, the research team first cracked the highly complex wild maize genome
.
Through the strategy of combining three-generation sequencing technology and three-dimensional genome, they explored and successfully assembled a hybrid and complex wild maize haploid genome (Zea mays ssp.
parviglumis, accession number Ames21814) for the localization and cloning
of wild maize high-protein genes.
After painstaking research, the research team created more than 10 generations of genetic material in a row, and finally constructed a high-generation near-generation gene line population of wild maize and common maize inbred line
B73.
In this process, they extracted DNA from more than 40,000 samples for genotyping, determined the protein content of more than 20,000 samples for phenotypic analysis, and backbred the population in the 4th generation BC4(n=500), 6th generation BC6(n=1314) and the 8th generation BC8(n=1344) performed three large-scale sequencing of high-protein genetic populations and fine map cloning, and finally cloned from wild maize to the first main gene THP9
to control the high protein content of maize.
This gene encodes asparagine synthetase 4 (ASN4), the center of nitrogen metabolism responsible for the synthesis of asparagine
.
Asparagine plays a central role in the nitrogen cycle and acts as a nitrogen donor in intermolecular transfer reactions of amino groups
.
Therefore, asparagine levels in plants are closely related
to seed protein content.
The study found that the expression of Thp9-T was significantly higher in the excellent gene Thp9-T in wild maize, while B73 and some maize inbred lines contained Thp9, a mutant form of Thp9, resulting in lower expression of ASN4
.
After the introduction of wild maize excellent gene Thp9-T into maize inbred line B73, the protein content of seeds increased by about 35%, the nitrogen content in roots increased by about 54%, the nitrogen content in stems increased by about 94%, the nitrogen content in leaves increased by about 18%, and the biomass, that is, the overall weight of plants, also increased
greatly.

In addition, the research team conducted large-scale field experiments in the Sanya Nanfan Base, and introduced the wild maize high-protein gene Thp9-T hybrid into Zhengdan 958, the maize production cultivar, which has the largest popularized area in China, which can significantly increase the protein content of hybrid seeds, indicating that this gene has important application potential
in cultivating high-protein maize.
At the same time, under the condition of reducing nitrogen fertilizer application, the biomass of maize and the nitrogen content level in plants and grains can be effectively maintained, which is of great significance
for promoting high yield and stable yield of maize under low nitrogen conditions.

In this study, a key excellent mutant gene Thp9-T that controls the formation of high-protein maize was found in wild maize, which can improve the assimilation efficiency of nitrogen in maize and thus facilitate the production of more protein
.
The introduction of Thp9-T into modern maize varieties greatly improved amino acid levels, especially asparagine, and increased seed protein content
without affecting grain weight.
At the same time, in the field experiment, this study also verified that Thp9-T plays a major role in the process of high-protein breeding improvement, which not only significantly increases the grain protein content of maize cultivars Zhengdan 958, but also effectively maintains the biomass of maize and the nitrogen content level of plants and grains under low nitrogen conditions, laying a solid foundation
for the further promotion and application of this gene in the future.

Due to the overuse of chemical fertilizers, the excellent gene Thp9-T of wild maize is not subject to selection pressure
in the long-term breeding process.
This study not only successfully cloned the wild maize variant gene Thp9-T, which is conducive to the genetic improvement of modern cultivated maize to increase grain protein content, but also has important guiding significance for reducing fertilizer application and protecting the ecological environment in the future, and provides new solutions
for the construction and implementation of national food security strategy under the new situation, ensuring national food security and effective supply of important agricultural products, and promoting sustainable agricultural development.

Postdoctoral fellow Huang Yongcai, Associate Professor Wang Haihai, and PhD student Yidong Zhu of the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences are co-first authors of this paper, and Professor Wu Yongrui and Professor Wang Wenqin of Shanghai Normal University are co-corresponding authors
of this paper 。 Huang Xing, Ma Guangjin, Xiao Qiao, doctoral students of the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences, Wang Qiong, senior experimentalist, and Wang Jiechen, associate researcher; Li Shuai, Wu Xingguo, Jin Yongbo, Cui Yahui, master students of Shanghai Normal University; Professor Lu Xiaoduo and Qin Li Young Teacher of Qilu Normal University; Professor Liu Hongjun, Associate Professor Yang Xuerong, and Master Candidate Zhao Yao of Shandong Agricultural University; Bao Zhigui, Shenzhen Institute of Genomics; Academician Brian A.
Larkins of the University of Arizona also participated
.
This research was supported
by the Pilot B Project of the Chinese Academy of Sciences, the National Natural Science Foundation of China, the China Postdoctoral Science Foundation, and the Shanghai "Super Postdoc" Incentive Program.

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This remarkable work unravels the genetics of corn protein content and free amino acid accumulation through experiments in molecular biology, biochemistry, comparative genomics, quantitative genetics
, and breeding.
The authors identified a target in the wild relatives of maize, Cymbidium chinensis, that is expected to improve the protein content and nitrogen use efficiency of maize
.
This work demonstrates the great potential
of harnessing the wild relatives of crops to achieve sustainable agriculture.

This represents an impressive piece of work in which one of the sites associated with the high protein content of the seed was identified
.
The site was pinpointed to the THP9 gene, which boosted NUE and seed protein content
when introduced into modern maize.
This work highlights the potential to introduce genetic variation from wild ancestors into quality crops to promote more sustainable agriculture and future food security
.

THP9 in wild maize improves corn protein content and nitrogen efficiency