近日，華中農(nóng)業(yè)大學水產(chǎn)學院高澤霞教授課題組在Aquaculture期刊上在線發(fā)表了題為“Generation of Megalobrama amblycephala without intermuscular bones by runx2b gene mutation”的研究論文。該研究獲得了肌間刺完全缺失且生長發(fā)育正常的團頭魴F1代突變個體，不僅為獲得可穩(wěn)定遺傳的團頭魴新品種奠定了重要基礎，也為無肌間刺性狀從模式物種到經(jīng)濟魚類上的應用提供了很好的示例。

 

　　肌間刺起源于肌腱祖細胞，膜內(nèi)骨化而來，僅存在于低等真骨魚類中。世界主要養(yǎng)殖魚類都含有一定數(shù)量的肌間刺，特別是我國主養(yǎng)的青草鰱鳙鯉鯽魴等大宗淡水魚類，食用時極易出現(xiàn)卡喉嚨的危險，嚴重影響了其水產(chǎn)品的經(jīng)濟價值。課題組前期以模式魚類斑馬魚為研究材料，篩選鑒定出調(diào)控魚類肌間刺發(fā)生的關鍵基因runx2b，2022年發(fā)表于National Science Reivew上。接著，課題組開始探索是否可以將這一模式物種上的研究發(fā)現(xiàn)應用到經(jīng)濟魚類上。

 

　　課題組以長期開展遺傳選育工作的團頭魴（俗稱武昌魚）為經(jīng)濟養(yǎng)殖魚類代表開展驗證工作。團頭魴屬于鯉形目、鯉科、鲌亞科、魴屬魚類，是我國重要的淡水養(yǎng)殖經(jīng)濟魚類，約含118根肌間刺。課題組前期已通過分子輔助選育技術培育出生長速度快、成活率高的團頭魴“華海1號”國審新品種，在該品種的基礎上，采用基因編輯獲得肌間刺部分缺失的團頭魴突變體F0代（肌間刺缺失5.6%–33.8%），進一步將F0代雌雄突變個體進行人工繁殖獲得F1代。課題組隨機篩選45天的團頭魴F1代品系60條，運用茜素紅整體骨骼染色方法觀察團頭魴F1代肌間刺的表型，結(jié)果顯示27條團頭魴的肌間刺完全缺失。在F1代群體4月齡時，課題組利用便攜式X光掃描儀在5000尾個體中篩選到了316尾完全沒有肌間刺的團頭魴個體，可作為2023年繁殖無肌間刺團頭魴的親本。同時，研究發(fā)現(xiàn)完全無肌間刺團頭魴F1代幼魚個體骨骼的礦化、肌肉脂肪酸和氨基酸含量等性狀均未受到顯著影響。目前，無肌間刺團頭魴生長性狀良好，6月齡無刺團頭魴平均體重達到了150g以上，部分個體已達到250 g。課題組后期將進一步跟蹤研究無肌間刺團頭魴成魚的骨骼礦化、肌肉營養(yǎng)成分等性狀差異情況。

 

　　華中農(nóng)業(yè)大學水產(chǎn)學院碩士生董強和博士后聶春紅為論文共同第一作者，水產(chǎn)學院高澤霞教授為論文通訊作者。該研究得到國家大宗淡水魚類產(chǎn)業(yè)技術體系、湖北洪山實驗室、國家自然科學基金、國家重點研發(fā)計劃等項目的支持。

 

　　【英文摘要】

 

　　Blunt snout bream （Megalobrama amblycephala） is an important herbivorous freshwater economic fish cultivated in China. Its economic value has been affected by the existence of intermuscular bones （IBs）。 Our previous study proved that runx2b is the key gene for the IBs formation, and lack of runx2b gene could obtain a stable zebrafish mutant without IBs. We found runx2b mutant F0 generation of blunt snout bream through CRISPR/Cas9 editing technology had a significant reduction on the number of IBs. In order to further obtain the blunt snout bream without IBs, we used the runx2b mutant F0 generation to self-bred to obtain F1 generation. Subsequently, the 35 runx2b mutant F1 individuals of blunt snout bream without IBs were genotyped and found the number of runx2b targeting sites 1, 2 and 3 obtained 3 （1 insertion, 2 deletion）， 13 （8 insertion, 5 deletion） and 7 （1 insertion, 6 deletion） mutation types with the mutation efficiency being 25.71%, 94.28%, and 71.43%, respectively. Alizarin red bone staining and X-ray scan analysis showed that IBs were not existed in runx2b mutant F1 individuals, and micro-CT analysis found that the mineralization of other bone in runx2b mutant was not significantly affected compared to wild type fish （P > 0.05）。 RT-qPCR results also exhibited that bone-related genes had a lower expression level in runx2b mutants compared to wild type fish. Subsequently, we used the national standard methods to detect nutrition composition of muscle tissue in wild type （with IBs） and runx2b mutant F1 individuals （without IBs） at 120 dpf. The results showed that the basic muscle nutritional composition （moisture, crude protein, crude lipid and crude ash）， and the contents of most amino acids and fatty acids had no significant differences between wild and runx2b mutant at juvenile stage （P > 0.05）。 This study proves that the runx2b gene has an essential role for IBs formation in aquaculture species and lays an important foundation for obtaining a new blunt snout bream strain without IBs, which provides a good example for the application of IBs-free characters from model fish to commercial fish.

 

　　原文鏈接：https://doi.org/10.1016/j.aquaculture.2023.739263

 

　　Blunt snout bream （Megalobrama amblycephala） is an important herbivorous freshwater economic fish cultivated in China. Its economic value has been affected by the existence of intermuscular bones （IBs）。 Our previous study proved that runx2b is the key gene for the IBs formation, and lack of runx2b gene could obtain a stable zebrafish mutant without IBs. We found runx2b mutant F0 generation of blunt snout bream through CRISPR/Cas9 editing technology had a significant reduction on the number of IBs. In order to further obtain the blunt snout bream without IBs, we used the runx2b mutant F0 generation to self-bred to obtain F1 generation. Subsequently, the 35 runx2b mutant F1 individuals of blunt snout bream without IBs were genotyped and found the number of runx2b targeting sites 1, 2 and 3 obtained 3 （1 insertion, 2 deletion）， 13 （8 insertion, 5 deletion） and 7 （1 insertion, 6 deletion） mutation types with the mutation efficiency being 25.71%, 94.28%, and 71.43%, respectively. Alizarin red bone staining and X-ray scan analysis showed that IBs were not existed in runx2b mutant F1 individuals, and micro-CT analysis found that the mineralization of other bone in runx2b mutant was not significantly affected compared to wild type fish （P > 0.05）。 RT-qPCR results also exhibited that bone-related genes had a lower expression level in runx2b mutants compared to wild type fish. Subsequently, we used the national standard methods to detect nutrition composition of muscle tissue in wild type （with IBs） and runx2b mutant F1 individuals （without IBs） at 120 dpf. The results showed that the basic muscle nutritional composition （moisture, crude protein, crude lipid and crude ash）， and the contents of most amino acids and fatty acids had no significant differences between wild and runx2b mutant at juvenile stage （P > 0.05）。 This study proves that the runx2b gene has an essential role for IBs formation in aquaculture species and lays an important foundation for obtaining a new blunt snout bream strain without IBs, which provides a good example for the application of IBs-free characters from model fish to commercial fish.